Method for producing transgenic plants resistant to weed control compounds which disrupt the porphyrin pathways of plants

ABSTRACT

Methods for producing a transgenic plant that is resistant to a weed control compound including introducing a nucleotide sequence encoding a coproporphyrinogen III oxidase of  Escherichia coli  into a plant cell, expressing the nucleotide sequence within the plant cell, and regenerating the plant cell into a transgenic plant and transgenic plants produced by such methods. Methods for controlling weeds including applying a weed control compound to an area containing transgenic plants containing a nucleotide sequence encoding a coproporphyrinogen III oxidase of  Escherichia coli . Methods for selecting plants or plant cells resistant to a weed control compound including applying a weed control compound to transgenic plants or transgenic plant cells containing a nucleotide sequence encoding a coproporphyrinogen III oxidase of  Escherichia coli.

BACKGROUND OF THE INVENTION

This application is a Divisional of Application No. 11/113,224 filed onApr. 25, 2005, which is a Divisional of U.S. application Ser. No.09/697,719, filed on Oct. 27, 2000, which issued on Jun. 14, 2005, asU.S. Pat. No. 6,906,245, which is a Continuation-in-part of U.S.application Ser. No. 09/302,357, filed on Apr. 30, 1999, which issued onMay 27, 2003, as U.S. Pat. No. 6,570,070, and for which priority isclaimed under 35 U.S.C. §120; and this application claims priority under35 U.S.C. §119 of Japanese Application Nos. JP 120553/1998, 281127/1998,330981/1998, and 054730/1999 filed in Japan on Apr. 30, 1998, Oct. 2,1998, Nov. 20, 1998, and Mar. 2, 1999, respectively. The entire contentsof all are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a method for giving resistance to weedcontrol compounds to plants.

DISCLOSURE OF THE RELATED ART

Weed control is very important work for improving yields and quality ofcultivated plants. For this purpose, weed control compounds such asherbicides are mainly used. However, for using weed control compounds,it is not always easy to distinguish cultivated plants from weeds ofallied species to selectively control only weeds. Then, production ofplants having resistance to weed control compounds (hereinafter referredto as weed control compound-resistance) has been attempted and someresistant plants have been put to practical use.

Recently, gene engineering techniques have been utilized for producingplants having weed control compound-resistance. As such a technique, forexample, Hinchee, M. A. W. et al. disclose a method for producing aplant having resistance to a herbicide, glyphosate, wherein5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene which is atarget enzyme of glyphosate is mutagenized so that an affinity forglyphosate is reduced, and the gene is introduced into a plant [Hinchee,M. A. W. et al., BIO/TECHNOLOGY, 6: p 915 (1988)].

OBJECT OF THE INVENTION

Varieties of known methods for giving weed control compound-resistanceto plants are not necessarily sufficient and it has been desired todevelop further various kinds of methods for giving weed controlcompound-resistance to plants.

The main object of the present invention is to provide a new kind of amethod for giving weed control compound-resistance to plants.

This object as well as other objects and advantages of the presentinvention will become apparent to those skilled in the art from thefollowing description with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the restriction map of plasmid pETBCH. bchH is magnesiumchelatase protoporphyrin IX binding subunit gene of a photosyntheticbacterium Rhodobacter sphaeroides. T7 pro represents the promotersequence of T7 phage, and T7 ter represents the terminator sequence ofT7 phage. Ampr is an ampicillin resistant gene, lacIq is a repressorprotein gene of a lactose operon, and ori is the replication origin.

FIG. 2 is the restriction map of plasmid pACYCSP. PPO isprotoporphyrinogen IX oxidase gene of soybean and lac pro represents thepromoter sequence of a lactose operon. Cmr is a chloramphenicolresistant gene and ori is the replication origin.

FIG. 3 is the restriction map of plasmid pTVBCH. bchH is magnesiumchelatase protoporphyrin IX binding subunit gene of the photosyntheticbacterium Rhodobacter sphaeroides. lac pro represents the promotersequence of a lactose operon. Ampr is an ampicillin resistant gene andori is the replication origin.

FIG. 4 is the restriction map of plasmid pBIBCH. bchH is magnesiumchelatase protoporphyrin IX binding subunit gene of the photosyntheticbacterium Rhodobacter sphaeroides. NP is the promoter sequence of anopaline synthase gene, NT is the terminator sequence of the nopalinesynthase gene, and 35S is the 35S promoter of cauliflower mosaic virus.NPTII represents a kanamycin resistant gene, and RB and LB representright and left border sequences of T-DNA, respectively.

FIG. 5 is the restriction map of plasmid pNO. NP is the promotersequence of a nopaline synthase gene, NT is the terminator sequence ofthe nopaline synthase gene, and 35S is the 35S promoter of cauliflowermosaic virus. NPTII represents a kanamycin resistant gene, and RB and LBrepresent right and left border sequences of T-DNA, respectively.

FIG. 6 is the restriction map of plasmid pTCHLH. TCHLH is protoporphyrinIX binding subunit gene of tobacco magnesium chelatase whose chloroplasttransit signal has been deleted, lac pro represents the promotersequence of a lactose operon. Ampr is an ampicillin resistant gene, Kmris a kanamycin resistant gene and ori is the replication origin.

FIG. 7 is the restriction map of plasmid pBITCHLH. TCHLH isprotoporphyrin IX binding subunit gene of tobacco magnesium chelatasewhose chloroplast transit signal has been deleted. NP is the promotersequence of a nopaline synthase, NT is the terminator sequence of thenopaline synthase and 35S is the 35S promoter of cauliflower mosaicvirus. NPTII represents a kanamycin resistant gene, and RB and LBrepresent right and left border sequences of T-DNA, respectively.

FIG. 8 is the restriction map of plasmid pTVGMP. GMP is soybeanprotoporphyrinogen IX oxidase gene whose chloroplast transit signal andFAD binding sequence have been deleted, lac pro represents the promotersequence of a lactose operon. Ampr represents an ampicillin resistantgene and ori is the replication origin.

FIG. 9 is the restriction map of plasmid pBIGMP. GMP is soybeanprotoporphyrinogen oxidase gene whose chloroplast transit signal and FADbinding sequence have been deleted. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and RB and LB are the right and left bordersequences of T-DNA, respectively.

FIG. 10 is the restriction map of plasmid pTVCRP. CRP isprotoporphyrinogen oxidase gene of Chlamydomonas reinhardtii whosechloroplast transit signal and FAD binding sequence have been deleted,lac pro represents the promoter sequence of a lactose operon. Ampr is anampicillin resistant gene and ori is the replication origin.

FIG. 11 is the restriction map of plasmid, pBICRP. CRP isprotoporphyrinogen oxidase gene of Chlamydomonas reinhardtii whosechloroplast transit signal and FAD binding sequence have been deleted.NP is the promoter sequence of a nopaline synthase, NT is the terminatorsequence of a nopaline synthase, and 35S is the 35S promoter ofcauliflower mosaic virus. NPTII is a kanamycin resistant gene, and RBand LB are the right and left border sequences of T-DNA, respectively.

FIG. 12 is the restriction map of plasmid pTVHVFI. HVF is barleyferrochelatase gene whose signal sequence has been deleted, lac prorepresents the promoter sequence of a lactose operon. Ampr represents anampicillin resistant gene and ori is the replication origin.

FIG. 13 is the restriction map of plasmid pBIHVF. HVF is barleyferrochelatase gene whose signal sequence has been deleted. NP is thepromoter sequence of a nopaline synthase, NT is the terminator sequenceof a nopaline synthase, and 35S is the 35S promoter of cauliflowermosaic virus. NPTII is a kanamycin resistant gene, and RB and LB are theright and left border sequences of T-DNA, respectively.

FIG. 14 is the restriction map of plasmid pTVCSF. CSF is cucumberferrochelatase gene whose signal sequence has been deleted, lac prorepresents the promoter sequence of a lactose operon. Ampr is anampicillin resistant gene, and ori is the replication origin.

FIG. 15 is the restriction map of plasmid pBICSF. CSF is cucumberferrochelatase gene whose signal sequence has been deleted. NP is thepromoter sequence of a nopaline synthase, NT is the terminator sequenceof a nopaline synthase, and 35S is the 35S promoter of cauliflowermosaic virus. NPTII is a kanamycin resistant gene, and RB and LB are theright and left border sequences of T-DNA, respectively.

FIG. 16 is the restriction map of plasmid pHEMF. HEMF iscoproporphyrinogen III oxidase gene (hemF) of Escherichia coli. lac prois the promoter sequence of a lactose operon. Ampr is an ampicillinresistant gene, and ori is the replication origin.

FIG. 17 is the restriction map of plasmid pBIHEMF. HEMF iscoproporphyrinogen III oxidase gene (hemF) of Escherichia coli. NP isthe promoter sequence of a nopaline synthase, NT is the terminatorsequence of a nopaline synthase, and 35S is the 35S promoter ofcauliflower mosaic virus. NPTII is a kanamycin resistant gene, and RBand LB are the right and left border sequences of T-DNA, respectively.

FIG. 18 is the restriction map of plasmid pBIHASYS8. HASYS8 is a geneencoding MG(HASYS)8 protein. NP is the promoter sequence of a nopalinesynthase, NT is the terminator sequence of a nopaline synthase, and 35Sis the 35S promoter of cauliflower mosaic virus. NPTII is a kanamycinresistant gene, and RB and LB are the right and left border sequences ofT-DNA, respectively.

FIG. 19 is the restriction map of plasmid pBIRASSL8. RASSL8 isMG(RASSL)8 protein. NP is the promoter sequence of a nopaline synthase.NT is the terminator sequence of a nopaline synthase. and 35S is the 35Spromoter of cauliflower mosaic virus. NPTII is a kanamycin resistantgene, and RB and LB are the right and left border sequences of T-DNA,respectively.

FIG. 20 is the restriction map of plasmid pNATP. PPO(A220V) is a PPOgene having a herbicidal compound-resistant mutation (A220V). NP is thepromoter sequence of a nopaline synthase. NT is the terminator sequenceof a nopaline synthase, and 35S is the 35S promoter of cauliflowermosaic virus. NPTII is a kanamycin resistant gene, and RB and LB are theright and left border sequences of T-DNA, respectively.

FIG. 21 is the restriction map of plasmid pBIAPTCH. PPO(A220V) is a PPOgene having a herbicidal compound-resistant mutation (A220V) and TCHLHis a tabacco magnesium chelatase subunit gene whose chloroplast transitsingal has been deleted. NP is the promoter sequence of a nopalinesynthase, NT is the terminator sequence of a nopaline synthase, and 35Sis the 35S promoter of cauliflower mosaic virus. NPTII is a kanamycinresistant gene, and RB and LB are the right and left border sequences ofT-DNA, respectively.

FIG. 22 is the restriction map of plasmid pCRATF. ATF is achloroplast-localized type ferrochelatase gene of Arabidopsis thaliana.lac pro represents the promoter sequence of a lactose operon. Ampr is anampicillin resistant gene, Kmr is kanamycin resistant gene and ori isthe replication origin.

FIG. 23 is the restriction map of plasmid pBIATF. ATF is achloroplast-localized type ferrochelatse gene of Arabidopsis thaliana.NP is the promoter sequence of a nopaline synthase. NT is the terminatorsequence of a nopaline synthase, and 35S is the 35S promoter ofcauliflower mosaic virus. NPTII is a kanamycin resistant gene, and RBand LB are the right and left border sequences of T-DNA, respectively.

FIG. 24 is the restriction map of plasmid pBIAPATF. PPO(A220V) is PPOgene having a herbicidal compound-resistant mutation (A220V). ATF is achloroplast-localized type ferrochelatase gene of Arabidopsis thaliana.NP is the promoter sequence of a nopaline synthase. NT is the terminatorsequence of a nopaline synthase, and 35S is the 35S promoter ofcauliflower mosaic virus. NPTII is a kanamycin resistant gene, and RBand LB are the right and left border sequences of T-DNA, respectively.

FIG. 25 is the restriction map of plasmid pCRSCPOX. SCPOX is soybeancoproporphyrinogen III oxidase gene and lac pro represents the promotersequence of a lactose operon. Ampr is an ampicillin resistant gene, Kmris a kanamycin resistant gene and ori is the replication origin.

FIG. 26 is the restriction map of plasmid pBISCPOX. SCPOX is soybeancoproporphyrinogen III oxidase gene. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and RB and LB are the right and left bordersequences of T-DNA, respectively.

FIG. 27 is the restriction map of plasmid pBIAPSCP. PPO(A220V) is PPOgene having a herbicidal compound-resistant mutation (A220V). SCPOX issoybean coproporphyrinogen III oxidase gene. NP is the promoter sequenceof a nopaline synthase, NT is the terminator sequence of a nopalinesynthase, and 35S is the 35S promoter of cauliflower mosaic virus. NPTIIis a kanamycin resistant gene, and RB and LB are the right and leftborder sequences of T-DNA, respectively.

FIG. 28 is the restriction map of plasmid pCREPSPS. CTP-EPSPS is avariant gene in which EPSPS gene derived from Agrobacterium is ligatedto the downstream of a nucleotide sequence encoding a chloroplasttransit peptide of EPSPS derived from petunia. lac pro represents thepromoter sequence of a lactose operon. Ampr is an ampicillin resistantgene, Kmr is kanamycin resistant gene and ori is the replication origin.

FIG. 29 is the restriction map of plasmid pNG01. NP is the promotersequence of a nopaline synthase, NT is the terminator sequence of anopaline synthase, and 35S is the 35S promoter of cauliflower mosaicvirus. NPTII is a kanamycin resistant gene, GUS is β-glucuronidase gene,and RB and LB are the right and left border sequences of T-DNA,respectively.

FIG. 30 is the restriction map of plasmid pNG04. NP is the promotersequence of a nopaline synthase, NT is the terminator sequence of anopaline synthase, and 35S is the 35S promoter of cauliflower mosaicvirus. NPTII is a kanamycin resistant gene, GUS is β-glucuronidase gene,and RB and LB are the right and left border sequences of T-DNA,respectively.

FIG. 31 is the restriction map of plasmid pNT35S. NT is the terminatorsequence of a nopaline synthase, 35S is the 35S promoter of cauliflowermosaic virus, and lac pro is the promoter sequence of a lactose operon.Ampr is an ampicillin resistant gene and ori is the replication origin.

FIG. 32 is the restriction map of plasmid pCENS. CTP-FPSPS is a variantgene in which EPSPS gene derived from Agrobacterium is ligated to thedownstream of a nucleotide sequence encoding a chloroplast transitpeptide of EPSPS derived from petunia. NT is the terminator sequence ofa nopaline synthase, 35S is the 35S promoter of cauliflower mosaicvirus, and lac pro is the promoter sequence of a lactose operon. Ampr isan ampicillin resistant gene, ori is the replication origin.

FIG. 33 is the restriction map of plasmid pCENSK. CTP-FPSPS is a variantgene in which EPSPS gene derived from Agrobacterium is ligated to thedownstream of a nucleotide sequence encoding a chloroplast transitpeptide of EPSPS derived from petunia. NT is the terminator sequence ofa nopaline synthase, 35S is the 35S promoter of cauliflower mosaicvirus, and lac pro is the promoter sequence of a lactose operon. Ampr isan ampicillin resistant gene, ori is the replication origin.

FIG. 34 is the restriction map of plasmid pBICE. CTP-EPSPS is a variantgene in which EPSPS gene derived from Agrobacterium is ligated to thedownstream of a nucleotide sequence encoding a chloroplast transitpeptide of EPSPS derived from petunia. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and PB and LB are the right and left bordersequences of T-DNA, respectively.

FIG. 35 is the restriction map of plasmid pBICETCH. CTP-EPSPS is avariant gene in which EPSPS gene derived from Agrobacterium is ligatedto the downstream of a nucleotide sequence encoding a chloroplasttransit peptide of EPSPS derived from petunia. TCHLH is protoporphyrinIX binding subunit gene of tabacco magnesium chelatase whose chloroplasttransit singal has been deleted. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and RB and LB are the right and left bordersequences of T-DNA, respectively.

FIG. 36 is the restriction map of plasmid pBIGMP. GMP is soybean PPOgene whose chloroplast transit signal and FAD binding sequence have beendeleted. NP is the promoter sequence of a nopaline synthase, NT is theterminator sequence of a nopaline synthase, and 35S is the 35S promoterof cauliflower mosaic virus. NPTII is a kanamycin resistant gene, and RBand LB are the right and left border sequences of T-DNA, respectively.

FIG. 37 is the restriction map of plasmid pBICEGMP. CTP-ESPSPS is achimera gene in which EPSPS gene derived from Agrobacterium is ligatedto the downstream of a nucleotide sequence encoding a chloroplasttransit peptide of EPSPS derived from petunia. GMP is soybean PPO genewhose chloroplast transit signal and FAD binding gene have been deleted.NP is the promoter sequence of a nopaline synthase, NT is the terminatorsequence of a nopaline synthase, and 35S is the 35S promoter ofcauliflower mosaic virus. NPTII is a kanamycin resistant gene, and RBand LB are the right and left border sequences of T-DNA, respectively.

FIG. 38 is the restriction map of plasmid pBICRP. CRP is PPO gene ofChlamydomonas reinhardtii whose chloroplast transit signal and FADbinding sequence have been deleted. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and RB and LB are the right and left bordersequences of T-DNA, respectively.

FIG. 39 is the restriction map of plasmid pBICECRP. CTP-EPSPS is avariant gene in which EPSPS gene derived from Agrobacterium is ligatedto the downstream of a nucleotide sequence encoding a chloroplasttransit peptide of EPSPS derived from petunia. CRP is PPO gene ofChlamydomonas reinhardtii whose chloroplast transit signal and FADbinding sequence have been deleted. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and RB and LB are the right and left bordersequences of T-DNA, respectively.

FIG. 40 is the restriction map of plasmid pBICEATF. CTP-EPSPS is avariant gene in which EPSPS gene derived from Agrobacterium is ligatedto the downstream of a nucleotide sequence encoding a chloroplasttransit peptide of EPSPS originated from petunia. ATF ischloroplast-localized type ferrochelatase gene of Arabidopsis thaliana.NP is the promoter sequence of a nopaline synthase, NT is the terminatorsequence of a nopaline synthase, and 35S is the 35S promoter ofcauliflower mosaic virus. NPTII is a kanamycin resistant gene, and RBand LB are the right and left border sequences of T-DNA, respectively.

FIG. 41 is the restriction map of plasmid pBISCPOX. SCPOX is soybeancoproporphyrinogen III oxydase gene. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and RB and LB are the right and left bordersequences of T-DNA, respectively.

FIG. 42 is the restriction map of plasmid pBICESCPOX. CTP-EPSPS is avariant gene in which EPSPS gene derived from Agrobacterium is ligatedto the downstream of a nucleotide sequence encoding a chloroplasttransit peptide of EPSPS derived from petunia. SCPOX is soybeancoproporphyrinogen III oxydase gene. NP is the promoter sequence of anopaline synthase, NT is the terminator sequence of a nopaline synthase,and 35S is the 35S promoter of cauliflower mosaic virus. NPTII is akanamycin resistant gene, and RB and LB are the right and left bordersequences of T-DNA, respectively.

BRIEF SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method for giving weedcontrol compound-resistance to a plant which comprises the steps of:

introducing a gene encoding a protein having the followingcharacteristics (a) to (c):

(a) having a specific affinity for a substance which is concerned withthe weed control activity of a weed control compound,

(b) having substantially no capability of modifying a substance forwhich said protein has a specific affinity, and

(c) being substantially free from framework regions of variable regionsin an immunoglobulin, into a plant cell; and

expressing the gene (hereinafter referred to as the first aspect of themethod of the present invention).

The present invention also relates to a method according to the above,wherein the gene is introduced into the plant cell in the form that itis operably ligated to a promoter and a terminator both of which arefunctional in the plant cell;

The method according to the above, wherein the substance which isconcerned with the weed control activity of the weed control compound isthe weed control compound itself;

The method according to the above, wherein the substance which isconcerned with the weed control activity of a weed control compound isan endogenous substance in a plant;

The method according to the above, wherein the weed control compound isthat inhibiting porphyrin biosynthesis of a plant;

The method according to the above, wherein the weed control compound isa protoporphyrinogen IX oxidase inhibitory-type herbicidal compound;

The method according to the above, wherein the substance which isconcerned with the weed control activity of the weed control compound isprotoporphyrin IX;

The method according to the above, wherein the protein is protoporphyrinIX binding subunit protein of magnesium chelatase, or a variant of saidprotein having a specific affinity for protoporphyrin IX;

The method according to the above, wherein the protein is magnesiumchelatase derived from a photosynthetic microorganism;

The method according to the above, wherein the protein is magnesiumchelatase derived from a plant;

The method according to the above, wherein the protein is magnesiumchelatase derived from tobacco;

The method according to the above, wherein the protein comprises theamino acid sequence of SEQ ID NO: 53;

The method according to the above, wherein the protein has the aminoacid sequence of SEQ ID NO: 54;

The method according to the above, wherein the protein comprises theamino acid sequence of SEQ ID NO: 55;

The method according to the above, wherein the protein has the aminoacid sequence of SEQ ID NO: 56;

The method according to the above, wherein the protein comprises theamino acid sequence of SEQ ID NO: 57;

The method according to the above, wherein the protein has the aminoacid sequence of SEQ ID NO: 58;

The method according to the above, wherein the protein comprises theamino acid sequence of SEQ ID NO: 59;

The method according to the above, wherein the protein has the aminoacid sequence of SEQ ID NO: 60;

The method according to the above, wherein the protein is composed of 4to 100 amino acids;

The method according to the above, wherein the substance which isconcerned with the weed control activity of the weed control compound isprotoporphyrinogen IX;

The method according to the above, wherein the protein is a variant ofprotoporphyrinogen IX oxidase having no capability of oxidizingprotoporphyrinogen IX and having a specific affinity for aprotoporphyrinogen IX;

The method according to the above, wherein the protein is a variant ofprotoporphyrinogen IX oxidase having no capability of oxidizingprotoporphyrinogen IX and having a specific affinity for aprotoporphyrin IX oxidase inhibitory-type herbicidal compound;

The method according to the above, wherein the protein is a variant ofprotoporphyrinogen IX oxidase derived from a plant;

The method according to the above, wherein the protein is a variant ofprotoporphyrinogen IX oxidase derived from soybean;

The method according to the above, wherein the protein is a variant ofprotoporphyrinogen IX oxidase derived from an algae; and

The method according to the above, wherein the protein is a variant ofprotoporphyrinogen IX oxidase derived from Chlamydomonas.

Another aspect of the present invention relates to a method for givingweed control compound-resistance to a plant which comprises the stepsof:

introducing a gene encoding a protein having the followingcharacteristics (a) to (c):

(a) having a specific affinity for protoporphyrin IX,

(b) having substantially no capability of modifying protoporphyrinogenIX, and

(c) being substantially free from framework regions of variable regionsin an immunoglobulin, into a plant cell; and

expressing the gene (hereinafter referred to as the second aspect of themethod of the present invention).

The present invention also relates to a method according to the above,wherein the gene is introduced in the plant cell in the form that it isoperably ligated to a promoter and a terminator both of which arefunctional in the plant cell;

The method according to the above, wherein the weed control compound isthat inhibiting porphyrin biosynthesis of a plant;

The method according to the above, wherein the weed control compound isa protoporphyrinogen IX oxidase inhibitory-type herbicidal compound;

The method according to the above, wherein the protein is magnesiumchelatase or a variant of said protein having a specific affinity forprotoporphyrin IX;

The method according to the above, wherein the protein is ferrochelataseor a variant of said protein having an specific affinity forprotoporphyrin IX;

The method according to the above, wherein the protein is ferrochelatasederived from a plant;

The method according to the above, wherein the protein is ferrochelatasederived from barley;

The method according to the above, wherein the protein is ferrochelatasederived from cucumber; and

The method according to the above, wherein the protein is a peptidecomposed of 4 to 100 amino acids.

Another aspect of the present invention relates to a method for givingweed control compound-resistance to a plant which comprises the stepsof:

introducing a gene encoding a protein having the followingcharacteristics (a) to (c):

(a) having a specific affinity for protoporphyrinogen IX,

(b) having the capability for modifying coproporphyrinogen III, and

(c) being substantially free from framework regions of variable regionsin an immunoglobulin, into a plant cell; and

expressing the gene (hereinafter referred to as the third aspect of themethod of the present invention).

The present invention also relates to the method according to the above,wherein the gene is introduced into the plant cell in the form that itis operably ligated to a promoter and a terminator both of which arefunctional in the plant cell;

The method according to the above, wherein the protein iscoproporphyrinogen III oxidase or a variant of said protein having aspecific affinity for protoporphyrinogen IX;

The method according to the above, wherein the protein iscoproporphyrinogen III oxidase derived from a microorganism;

The method according to the above, wherein the protein iscoproporphyrinogen III oxidase derived from Escherichia coli;

A weed control compound-resistant plant whose resistance is given by themethod of the above;

A weed control compound-resistant plant whose resistance is given by themethod of the above;

A method for protecting a plant which comprises applying the weedcontrol compound to a growth area of the plant of the above;

A method for protecting a plant which comprises applying the weedcontrol compound to a growth area of the plant of the above;

A method for selecting a plant which comprises applying a weed controlcompound to which the plant of the above is resistant to a growth areaof the plant of the above and other plants, and selecting either planton the basis of difference in growth between the plants;

A method for selecting a plant which comprises applying a weed controlcompound to which the plant of the above is resistant to a growth areaof the plant of the above and other plants, and selecting either planton the basis of difference in growth between the plants;

The method according to the above, wherein the plants are plant cells;

The method according to the above, wherein the plants are plant cells;

The method according to the above, wherein the weed control compound isa protoporphyrinogen IX oxidase inhibitory-type herbicidal compoundselected from the compounds of (1) to (3) below, and the substance whichis concerned with the weed control activity of the weed control compoundis protoporphyrin IX, protoporphyrinogen IX or a protoporphyrinogen IXoxidase inhibitory-type herbicidal compound:

(1) chlormethoxynil, bifenox, chlornitrofen (CNP), acifluorfen(5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitorobenzoic acid) and itsethyl ester, acifluorfen-sodium, oxyfluorfen(2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-trifluoromethylbenzene),oxadiazon(3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxadiazol-2(3H)-one),2-[4-chloro-2-fluoro-5-(prop-2-ynyloxy)phenyl]-2,3,4,5,6,7-hexahydro-1H-isoindol-1,3-dione,chlorphthalim (N-(4-chlorophenyl)-3,4,5,6-tetrahydrophtalimide),TNPP-ethyl (ethyl2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate), orN3-(1-phenylethyl)-2,6-dimethyl-5-propyonylnicotinamide;

(2) a compound represented by the general formula: J-G (I), wherein G isa group represented by any one of the following general formulas G-1 toG-9 and J is a group represented by any one of the following generalformulas of J-1 to J-30:

wherein the dotted lines in the formulas J-5, J-6, J-12 and J-24represent that the left hand ring contains only single bonds, or onebond in the ring is a double bond between carbon atoms;

X is oxygen atom or sulfur atom;

Y is oxygen atom or sulfur atom;

R¹ is hydrogen atom or halogen atom;

R² is hydrogen atom, C₁-C₈alkyl group, C₁-C₈ haloalkyl group, halogenatom, OH group, OR²⁷ group, SH group, S(O)_(p)R²⁷ group, COR²⁷ group,CO₂R²⁷ group, C(O)SR²⁷ group, C(O)NR²⁹R³⁰ group, CHO group, CR²⁷═NOR³⁶group, CH═CR³⁷CO₂R₂₇ group, CH₂CHR³⁷CO₂R²⁷ group, CO₂N═CR³¹R³² group,nitro group, cyano group. NHSO₂R³³ group, NHSO₂NHR³³ group. NR²⁷R³⁸group. NH2 group or phenyl group optionally substituted with one or moreand the same or different C₁-C₄ alkyl groups;

p is 0, 1 or 2;

R³ is C₁-C₂ alkyl group, C₁-C₂ haloalkyl group, OCH₃ group, SCH₃ group,OCHF₂ group, halogen atom, cyano group or nitro group;

R⁴ is hydrogen atom, C₁-C₃ alkyl group, C₁-C₃ haloalkyl group or halogenatom;

R⁵ is hydrogen atom, C₁-C₃ alkyl group, halogen atom, C₁-C₃haloalkylgroup, cyclopropyl group, vinyl group, C₂ alkynyl group, cyano group,C(O)R³⁸ group, CO₂R³⁸ group, C(O)NR³⁸R³⁹ group, CR³⁴R³⁵CN group,CR³⁴R³⁵C(O)R³⁸ group, CR³⁴R³⁵CO₂R³⁸ group, CR³⁴R³⁵C(O)NR³⁸R³⁹ group,CHR³⁴OH group, CHR³⁴OC(O)R³⁸ group or OCHR³⁴OC(O)NR³⁸R³⁹ group, or, whenG is G-2 or G-6, R⁴ and R⁵ may form C═O group together with the carbonatom to which they are attached;

R⁶ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₂-C₆alkoxyalkyl group,C₃-C₆ alkenyl group or C₃-C₆ alkynyl group;

X¹ is single bond, oxygen atom, sulfur atom, NH group, N(C₁-C₃ alkyl)group, N(C₁-C₃ haloalkyl) group or N(allyl) group;

R⁷ is hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, halogenatom, S(O)₂(C₁-C₆alkyl) group or C(═O)R⁴⁰ group;

R⁸ is hydrogen atom, C₁-C₈ alkyl group, C₃-C₈ cycloalkyl group, C₃-C₈alkenyl group, C₃-C₈ alkynyl group, C₁-C₈ haloalkyl group, C₂-C₈alkoxyalkyl group, C₃-C₈ alkoxyalkoxyalkyl group, C₃-C₈haloalkynylgroup, C₃-C₈ haloalkenyl group, C₁-C₈ alkylsulfonyl group, C₁-C₈haloalkylsulfonyl group, C₃-C₈ alkoxycarbonylalkyl group, S(O)₂NH(C₁-C₈alkyl) group, C(O)R⁴¹ group or benzyl group whose phenyl ring may besubstituted with R⁴²;

n and m are independently 0, 1, 2 or 3 and m+n is 2 or 3;

Z is CR⁹R¹⁰ group, oxygen atom, sulfur atom, S(O) group, S(O)₂ group orN(C₁-C₄ alkyl) group;

each R⁹ is independently hydrogen atom, C₁-C₃ alkyl group, halogen atom,hydroxyl group, C₁-C₆ alkoxy group, C₁-C₆ haloalkyl group, C₁-C₆haloalkoxy group, C₂-C₆ alkylcarbonyloxy group or C₂-C₆haloalkylcarbonyloxy group;

each R¹⁰ is independently hydrogen atom, C₁-C₃ alkyl group, hydroxylgroup or halogen atom;

R¹¹ and R¹² are independently hydrogen atom, halogen atom, C₁-C₆ alkylgroup, C₃-C₆ alkenyl group or C₁-C₆ haloalkyl group;

R¹³ is hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₃-C₆alkenyl group, C₃-C₆ haloalkenyl group, C₃-C₆ alkynyl group, C₃-C₆haloalkynyl group, HC(═O) group, (C₁-C₄ alkyl)C(═O) group or NH₂group;

R¹⁴ is C₁-C₆ alkyl group, C₁-C₆ alkylthio group, C₁-C₆haloalkyl group orN(CH₃)₂ group;

W is nitrogen atom or CR¹⁵;

R¹⁵ is hydrogen atom, C₁-C₆ alkyl group, halogen atom, or phenyl groupoptionally substituted with C₁-C₆ alkyl group, one or two halogen atoms,C₁-C₆ alkoxy group or CF₃ group;

each Q is independently oxygen atom or sulfur atom;

Q¹ is oxygen atom or sulfur atom;

Z¹ is CR¹⁶R¹⁷ group, oxygen atom, sulfur atom. S(O) group, S(O)₂ groupor N(C₁-C₄alkyl) group;

each R¹⁶ is independently hydrogen atom, halogen atom, hydroxyl group,C₁-C₆ alkoxy group, C₁-C₆ haloalkyl group. C₁-C₆ haloalkoxy group, C₂-C₆alkylcarbonyloxy group or C₂-C₆ haloalkylcarbonyloxy group;

each R¹⁷ is independently hydrogen atom, hydroxyl group or halogen atom;

R¹⁸ is C₁-C₆ alkyl group, halogen atom or C₁-C₆ haloalkyl group;

R¹⁹ and R²⁰ are independently hydrogen atom, C₁-C₆ alkyl group, or C₁-C₆haloalkyl group;

Z² is oxygen atom, sulfur atom, NR⁹ group or CR⁹R¹⁰ group;

R²¹ and R²² are independently C₁-C₆ alkyl group, C₁-C₆ haloalkyl group,C₃-C₆ alkenyl group, C₃-C₆ haloalkenyl group, C₃-C₆ alkynyl group orC₃-C₆ haloalkynyl group;

R²³ is hydrogen atom, halogen atom or cyano group;

R²⁴ is C₁-C₆ alkylsulfonyl group, C₁-C₆ alkyl group, C₁-C₆ haloalkylgroup, C₃-C₆ alkenyl group, C₃-C₆ alkynyl group, C₁-C₆ alkoxy group,C₁-C₆ haloalkoxy group or halogen atom;

R²⁵ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₃-C₆ alkenyl group orC₃-C₆ alkynyl group;

R²⁶ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group or phenyl groupoptionally substituted with C₁-C₆ alkyl, one or two halogen atoms, oneor two nitro groups, C₁-C₆ alkoxy group or CF³ group;

W¹ is nitrogen atom or CH group;

T is a group represented by any one of the following general formulasT-1, T-2 and T-3:

(wherein E¹, E², E³, E⁴, E⁵, E⁶, E⁷, E⁸, E⁹, E¹⁰, E¹¹ and E¹² areindependently hydrogen atom or C₁-C₃ alkyl group);

R²⁷ is C₁-C₈ alkyl group. C₃-C₈ cycloalkyl group. C₃-C₈ alkenyl group,C₃-C₈alkynyl group, C₁-C₈ haloalkyl group, C₂-C₈ alkoxyalkyl group,C₂-C₈ alkylthioalkyl group, C₁-C₈ alkylsulfinylalkyl group, C₂-C₈alkylsulfonylalkyl group, C₁-C₈ alkylsulfonyl group, phenylsulfonylgroup whose phenyl ring may be substituted with at least one substituentselected from the group consisting of halogen atom and C₁-C₄ alkylgroup, C₄-C₈ alkoxyalkoxyalkyl group, C₄-C₈ cycloalkylalkyl group, C₆-C₈cycloalkoxyalkyl group, C₄-C₈ alkenyloxyalkyl group, C₄-C₈alkynyloxyalkyl group, C₃-C₈ haloalkoxyalkyl group, C₄-C₈haloalkenyloxyalkyl group, C₄-C₈ haloalkynyloxyalkyl group, C₆-C₈cycloalkylthioalkyl group, C₄-C₈ alkenylthioalkyl group, C₄-C₈alkynylthioalkyl group, C₁-C₄ alkyl group substituted with phenoxy groupwhose ring is substituted with at least one substituent selected fromthe group consisting of halogen atom, C₁-C₃ alkyl group and C₁-C₃haloalkyl group, benzyloxy group whose ring is substituted with at leastone substituent selected from the group consisting of halogen atom,C₁-C₃ alkyl group and C₁-C₃ haloalkyl group, C₄-C₈ trialkylsilylalkylgroup, C₃-C₈ cyanoalkyl group, C₃-C₈ halocycloalkyl group. C₃-C₈haloalkenyl group, C₅-C₈ alkoxyalkenyl group, C₅-C₈ haloalkoxyalkenylgroup, C₅-C₈ alkylthioalkenyl group, C₃-C₈ haloalkynyl group. C₅-C₈alkoxyalkynyl group, C₅-C₈ haloalkoxyalkynyl group, C₅-C₈alkylthioalkynyl group, C₂-C₈ alkylcarbonyl group, benzyl group whosering is substituted with at least one substituent selected from thegroup consisting of halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkylgroup, CHR³⁴COR²⁸ group, CHR³⁴COOR²⁸ group. CHR³⁴P(O)(OR²⁸)₂ group,CHR³⁴P(S)(OR²⁸)₂ group, CHR³⁴C(O)NR²⁹R³⁰ group or CHR³⁴C(O)NH² group;

R²⁸ is C₁-C₆ alkyl group, C₂-C₆ alkenyl group, C₃-C₆ alkynyl group ortetrahydrofuranyl group;

R²⁹ and R³¹ are independently hydrogen atom or C₁-C₄ alkyl group;

R³⁰ and R³² are independently C₁-C₄ alkyl group or phenyl group whosering may be substituted with at least one substituent selected from thegroup consisting of halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkylgroup; or,

R²⁹ and R³⁰ together may form —(CH₂)₅—, —(CH₂)₄— or —CH₂CH₂OCH₂CH₂—, orthe ring thus formed may be substituted with at least one substituentselected from the group consisting of C₁-C₃ alkyl group, phenyl groupand benzyl group; or,

R³¹ and R³² may from C₃-C₈ cycloalkyl group together with the carbonatom to which they are attached;

R³³ is C₁-C₄ alkyl group, C₁-C₄ haloalkyl group or C₃-C₆ alkenyl group;

R³⁴ and R³⁵ are independently hydrogen atom or C₁-C₄ alkyl group;

R³⁶ is hydrogen atom, C₁-C₆ alkyl group, C₃-C₆ alkenyl group or C₃-C₆alkynyl group;

R³⁷ is hydrogen atom, C₁-C₄ alkyl group or halogen atom;

R³⁸ is hydrogen atom. C₁-C₆ alkyl group, C₃-C₆ cycloalkyl group, C₃-C₆alkenyl group, C₃-C₆ alkynyl group, C₂-C₆ alkoxyalkyl group, C₁-C₆haloalkyl group, phenyl group whose ring may be substituted with atleast one substituent selected from the group consisting of halogenatom, C₁-C₄ alkyl group and C₁-C₄ alkoxy group, —CH₂CO₂(C₁-C₄ alkyl)group or —CH(CH₃)CO₂(C₁-C₄ alkyl) group;

R³⁹ is hydrogen atom, C₁-C₂ alkyl group or C(O)O(C₁-C₄ alkyl) group;

R⁴⁰ is hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ alkoxy group or NH(C₁-C₆alkyl) group;

R⁴¹ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₁-C₆ alkoxy group,NH(C₁-C₆ alkyl) group, phenyl group whose ring may be substituted withone substituent selected from the group consisting of R⁴² group, benzylgroup and C₂-C₈ dialkylamino group; and

R⁴² is C₁-C₆ alkyl group, one or two halogen atoms, C₁-C₆ alkoxy groupor CF₃ group;

(3) a compound of the formula (II):

or nipilacrofen,

wherein R⁴³ is C₁-C₄ alkyl group;

R⁴⁴ is C₁-C₄ alkyl group, C₁-C₄ alkylthio group. C₁-C₄ alkoxy group,C₁-C₄ haloalkyl group, C₁-C₄ haloalkylthio group or C₁-C₄ haloalkoxygroup;

R⁴³ and R⁴⁴ together may form —(CH₂)₃— or —(CH₂)₄-;

R⁴⁵ is hydrogen atom or halogen atom;

R⁴⁶ is hydrogen atom or C₁-C₄ alkyl group;

R⁴⁷ is hydrogen atom, nitro group, cyano group, —COOR⁴⁹ group,—C(═X)NR⁵⁰R⁵¹ group or —C(═X²)R⁵² group;

R⁴⁸ is hydrogen atom, halogen atom, cyano group, C₁-C₄ alkyl groupoptionally substituted with at least one substituent selected from thegroup consisting of halogen atom and hydroxyl group, C₁-C₄ alkoxy group,phenyl group optionally substituted with at least one substituentselected from the group consisting of halogen atom, nitro group, cyanogroup, C₁-C₄ alkyl group, C₁-C₄ alkoxy group and halo-C₁-C₄ alkyl group,pyrrolyl group, C₂-C₈ alkyl group, C₃-C₈ alkenyl group, C₃-C₈ alkynylgroup, C₃-C₈ alkoxy group, a group selected from the group consisting ofC₂-C₈ alkyl group, C₃-C₈ alkenyl group, C₃-C₈ alkynyl group and C₃-C₈alkoxy group into which at least one oxygen atom is inserted, or any oneof groups represented by the following formulas:

wherein R⁴⁹, R⁵⁰ and R⁵² are, the same or different, hydrogen atom orC₁-C₄ alkyl group;

R⁵⁰ and R⁵¹ may form saturated alicyclic 5 or 6 membered ring togetherwith the nitrogen atom to which they are attached;

R⁵² is hydrogen atom, C₁-C₄ alkyl group or C₁-C₄ alkyl group substitutedwith at least one halogen atom;

R⁵³ is hydrogen atom, C₁-C₄ alkyl group optionally substituted with atleast one halogen atom, C₂-C₆ alkenyl group optionally substituted withat least one halogen atom, C₃-C₆ alkynyl group optionally substitutedwith at least one halogen atom, phenyl group optionally substituted withat least one halogen atom, C₃-C₈ cycloalkyl group, cyanomethyl group, orR⁶³CO— group;

R⁵⁴ is hydrogen atom, C₁-C₆ alkyl group optionally substituted with atleast one halogen atom, C₂-C₆ alkenyl group optionally substituted withat least one halogen atom, C₃-C₆ alkynyl group optionally substitutedwith at least one halogen atom, phenyl group optionally substituted withhalogen atom, C₃-C₈ cycloalkyl group, cyanomethyl group, C₁-C₄alkoxy-C₁-C₆ alkyl group, di-C₁-C₄ alkylamino-C₁-C₄ alkyl group,tetrahydrofurfurylmethyl group, C₃-C₆ alkynyloxy-C₁-C₄ alkyl group,benzyl whose ring may be substituted with substituent selected from thegroup consisting of halogen atom, nitro group, cyano group, C₁-C₄ alkylgroup, C₁-C₄ alkoxy group and halo-C₁-C₄ alkyl group, —C(═X²)R⁶³ group,—(CH₂)a-(O)d-R⁷⁰ group, —(CH₂)a-O—(CH₂)b-R⁷⁰ group, —(CH₂)a-X^(2—R) ⁷⁶group;

R⁵³ and R⁵⁴ together with the nitrogen atom to which they are attachedmay form saturated alicyclic 3, 5 or 6 membered ring or aromatic 5 or 6membered ring in which a carbon atom may be optionally replaced withoxygen atom;

R⁵⁵ is hydrogen atom, C₁-C₄ alkyl group, C₂-C₆ alkenyl group or C₃-C₆alkynyl group, or R⁵⁵ and R⁵⁶ together may form —(CH₂)e-;

R⁵⁶ and R⁵⁷ are independently C₁-C₄ alkyl group optionally substitutedwith at least one halogen atom, C₂-C₆ alkenyl group optionallysubstituted with at least one halogen atom, C₃-C₆ alkynyl optionallysubstituted with at least one halogen atom or phenyl group optionallysubstituted with at least one halogen atom, hydrogen atom, C₃-C₆cycloalkyl group, —XR⁶⁰ group or —NR⁶¹R⁶² group;

R⁵⁸ is hydrogen atom, C₁C₆ alkyl group, C₂-C₆ alkenyl group, C₃-C₆alkynyl group, C₁-C₄ alkylcarbonyl group, cyano-C₁-C₃ alkyl group, C₁-C₄alkoxycarbonyl-C₁-C₄ alkyl group, di-C₁-C₄ alkoxycarbonyl-C₁-C₄ alkylgroup, benzyl group, C₁-C₄ alkoxy-C₁-C₄ alkynyl group, —(CH₂)a-R⁷⁵group, —(CH₂)a-X²—R⁷² group, —(CH₂)a-X²—(CH₂)b-R⁷² group or—(CH₂)a-X²—(CH₂)b-X²—(CH₂)c-R⁷² group;

R⁵⁹ is hydrogen atom, C₁-C₄ alkyl group, C₂-C₆ alkenyl group, C₃-C₆alkynyl group, cyano-C₁-C₃ alkyl group, C₁-C₄ alkylcarbonyl-C₁-C₃ alkylgroup or phenyl group;

R⁶⁰ is C₁-C₄ alkyl group optionally substituted with at least onehalogen atom;

R⁶¹ and R⁶² are, the same or different, hydrogen atom or C₁-C₄ alkylgroup;

R⁶³ is C₁-C₄ alkyl group optionally substituted with at least onehalogen atom, C₁-C₄ alkoxy-C₁-C₄ alkyl group, C₁-C₄ alkylthio-C₁-C₄alkyl group, C₃-C₆ cycloalkyl group, phenyl group whose ring may besubstituted with one substituent selected from the group consisting ofhalogen atom, nitro group, cyano group, C₁-C₄ alkyl group, C₁-C₄ alkoxygroup and halo-C₁-C₄ alkyl group, —NR⁷³R⁷⁴ group or —(CH₂)a-(O)d-R⁷⁵group;

R⁶⁴ is C₁-C₄ alkoxycarbonyl group or carboxyl group;

R⁶⁵ is chloromethyl group, cyanomethyl group, C₃-C₆ cycloalkyl groupinto which at least one oxygen atom may be inserted, or C₁-C₄alkoxycarbonyl-C₁-C₄ alkyl group;

R⁶⁶ is hydroxyl group or —NR⁶⁷R⁶⁸ group;

A is —NR⁶⁷R⁶⁸ group or —S(O)f-R⁶⁹ group;

R⁶⁷ and R⁶⁸ are, the same or different, hydrogen atom or C₁-C₄ alkylgroup;

R⁶⁹ is C₁-C₄ alkyl group or C₁-C₄ haloalkyl group;

R⁷⁰ is hydrogen atom, hydroxyl group, halogen atom, C₁-C₄ alkyl groupoptionally substituted with at least one C₁-C₄ alkoxy group, C₃-C₆cycloalkyl group into which at least one oxygen atom may be inserted,C₃-C₆ cycloalkyl group optionally substituted with one or two methylgroups, furyl group, thienyl group or —C(═O)R⁷¹ group;

R⁷¹ and R⁷² are, the same or different, C₁-C₄ alkyl group or C₁-C₄alkoxy group;

R⁷³ and R⁷⁴ are, the same or different, C₁-C₄ alkyl group or phenylgroup;

R⁷⁵ is C₃-C₆ cycloalkyl into which at least one oxygen atom may beinserted, C₃-C₆ cycloalkyl group optionally substituted with one or twomethyl groups, furyl group, thienyl group or —C(═O)R⁷¹ group;

R⁷⁶ is C₁-C₄ alkyl group;

a, b and c is independently 1, 2 or 3;

d is 0 or 1;

e is 2 or 3;

f is 1 or 2; and

X² is oxygen atom or sulfur atom.

The method according to the above, additionally comprising the steps of:

introducing into the plant cell, a second gene selected from a geneencoding a protein substantially having protoporphyrinogen oxidaseactivity, a gene encoding a protein substantially having5-enolpyruvylshikamate-3-phosphate synthase activity and a gene encodinga protein substantially having glyphosate oxidoreductase activity; and

expressing said second gene.

Another aspect of the present invention relates to a plant cell having:

a gene encoding a protein having the following characteristics (a) to(c):

(a) having a specific affinity for a substance which is concerned withthe weed control activity of a weed control compound,

(b) having substantially no capability of modifying a substance forwhich said protein has a specific affinity, and

(c) being substantially free from framework regions of variable regionsin an immunoglobulin; and

at least one altered form of an enzymatic activity which gives aresistance to a weed control compound in an amount inhibiting anaturally occurring form of said enzymatic activity, wherein saidaltered form of an enzymatic activity is a form of enzymatic activityselected from a protoporphyrinogen oxidase activity,5-enolpyruvylshikamate-3-phosphate synthase activity and glyphosateoxidoreductase activity.

Another aspect of the present invention relates to a plant cell having:

a gene encoding a protein having the following characteristics (a) to(c):

(a) having a specific affinity for a substance which is concerned withthe weed control activity of a weed control compound,

(b) having substantially no capability of modifying a substance forwhich said protein has a specific affinity, and

(c) being substantially tree from framework regions of variable regionsin an immunoglobulin; and an altered protoporphyrinogen oxidase activitywhich gives a resistance to a weed control compound in an amountinhibiting a natural occurring protopotphyrinogen oxidase activity.

Another aspect of the present invention relates to a plant cell having:

a gene encoding a protein having the following characteristics (a) to(c):

(a) having a specific affinity for a substance which is concerned withthe weed control activity of a weed control compound,

(b) having substantially no capability of modifying a substance forwhich said protein has a specific affinity, and

(c) being substantially free from framework regions of variable regionsin an immunoglobulin; and

an altered 5-enolpyruvylshikamate-3-phosphate synthase activity whichgives a resistance to a weed control compound in an amount inhibiting anatural occurring 5-enolpyruvylshikamate-3-phosphate synthase activity.

Another aspect of the present invention relates to a plant cell having:

a gene encoding a protein having the following characteristics (a) to(c):

(a) having a specific affinity for a substance which is concerned withthe weed control activity of a weed control compound,

(b) having substantially no capability of modifying a substance forwhich said protein has a specific affinity, and

(c) being substantially free from framework regions of variable regionsin an immunoglobulin; and

an altered glyphosate oxidoreductase activity which gives a resistanceto a weed control compound in an amount inhibiting a natural occurringglyphosate oxidoreductase activity.

The present invention also relates to the plant cell according to theabove, wherein said altered form of an enzymatic activity is conferredby a second gene selected from a gene encoding a protein substantiallyhaving a protoporphyrinogen oxidase activity, a gene encoding a proteinsubstantially having 5-enolpyruvylshikamate-3-phosphate synthaseactivity and a gene encoding a protein substantially having glyphosateoxidoreductase activity;

The plant cell according to the above, wherein the gene encoding aprotein having the following characteristics (a) to (c):

(a) having a specific affinity for a substance which is concerned withthe weed control activity of a weed control compound,

(b) having substantially no capability of modifying a substance forwhich said protein has a specific affinity, and

(c) being substantially free from framework regions of variable regionsin an immunoglobulin; and the second gene are introduced into the plantcell in the form in that both of said genes are operably ligated to apromoter and a terminator both of which are functional in said plantcell.

The plant cell according to the above, wherein the protein substantiallyhaving a proto-porphyrinogen IX oxidase activity is protoporphyrinogenIX oxidase, the protein substantially having a5-enolpyruvylshikamate-3-phosphate synthase activity is5-enolpyruvylshikamate-3-phosphate synthase and the proteinsubstantially having glyphosate oxidoreductase activity is glyphosateoxidoreductase;

The plant cell according to the above, wherein the plant cell is derivedfrom dicotyledones or monocotyledons;

A plant comprising the plant cell of the above;

A method for protecting a plant which comprises applying aprotoporphyrinogen IX oxidase inhibitory-type compound to a growth areaof the plant of the above;

A method for protecting a plant which comprises applying aprotoporphyrinogen IX oxidase inhibitory-type compound and a compoundinhibiting 5-enolpyruvylshikamate-3-phosphate synthase to a growth areaof the plant of the above;

A method for protecting a plant which comprises applying aprotoporphyrinogen IX oxidase inhibitory-type compound and a compoundinhibiting 5-enolpyruvylshikamate-3-phosphate synthase to a growth areaof the plant of the above;

A method for selecting a plant which comprises applying aprotoporphyrinogen IX oxidase inhibitory-type compound to a growth areaof the plant of the above and other plants, and selecting either planton the basis of difference in growth between the plants;

A method for selecting a plant which comprises applying aprotoporphyrinogen IX oxidase inhibitory-type compound and a compoundinhibiting 5-enolpyruvylshikamate-3-phosphate synthase to a growth areaof the plant of the above and other plants, and selecting either planton the basis of difference in growth between the plants; and

A method for selecting a plant which comprises applying aprotoporphyrinogen IX oxidase inhibitory-type compound and a compoundinhibiting 5-enolpyruvylshikamate-3-phosphate synthase to a growth areaof the plant of the above 63 and other plants, and selecting eitherplant on the basis of difference in growth between the plants.

DETAILED DESCRIPTION OF THE INVENTION

In the method of the present invention, substances which are concernedwith weed control activities of weed control compounds (hereinafterreferred to as weed control substances) are those constituting a part ofmetabolic reaction systems in organisms which are responsible for weedcontrol activities upon applying the compounds to plants. Examplesthereof include weed control compounds themselves, endogenous substancesin plants, and the like. Specifically, as such endogenous substances inplants, for example, there are substrates of target enzymes on whichweed control compounds act, or precursors or metabolites of thesubstrates which cause cellular dysfunction upon accumulating in plantcells; substances produced by the above substances in plant cells whichcause cellular dysfunction; and the like. More specifically, it has beenknown that, when a compound having herbicidal activity (hereinafterreferred to as herbicidal compound) which inhibits the activity ofprotoporphyrinogen IX oxidase (EC 1.3.3.4, hereinafter referred to asPPO) is applied to a plant, protoporphyrinogen IX which is the substrateof PPO is accumulated in the plant cells and it is metabolized to formprotoporphyrin X, followed by formation of active oxygen in the presenceof both protoporphyrin X and light in the cells, which damages cellfunctions [Junshi MIYAMOTO ed., Atarashii Noyaku no Kagaku (Chemistry ofNew Agrochemicals), Chapter 3, Section 3.3, p 106 ( 1993), HirokawaShoten, Tokyo]. Thus, protoporphyrinogen IX, protoporphyrin IX andactive oxygen in these systems, and the like can be exemplified as thesesubstances.

In the method of the present invention, weed control compounds includecompounds having herbicidal activities, plant growth regulatoractivities, and the like.

Examples of the herbicidal compounds include compounds inhibitingporphyrin biosynthesis, compounds inhibiting electron transfer inphotosynthesis, compounds inhibiting carotenoid biosynthesis, compoundsinhibiting amino acid biosynthesis, compounds inhibiting lipidbiosynthesis, compounds inhibiting cell wall biosynthesis, compoundsinfluencing protein biosynthesis, nucleic acid biosynthesis and celldivision, compounds having auxin antagonistic activity, and the like.More specifically, as the compounds inhibiting porphyrin biosynthesis,for example, there are compounds inhibiting PPO activity (PPOinhibitory-type herbicidal compound), and the like. As the compoundsinhibiting electron transfer in photosynthesis, for example, there arecompounds inhibiting electron transfer of photochemical system I or II,compounds inhibiting 4-hydroxyphenyl pyruvate dioxygenase (EC1.13.11.27; hereinafter referred to as 4-HPPD) which influencesbiosynthesis of plastoquinone which transfers electrons, and the like.As the compounds inhibiting carotenoid biosynthesis, for example, thereare compounds inhibiting phytoene desaturase (hereinafter referred to asPDS), and the like. As the compounds inhibiting amino acid biosynthesis,for example, there are compounds inhibiting EPSPS, acetolactate synthase(EC 4.1.3.18; hereinafter referred to as ALS), glutamine synthetase (EC6.3.1.2; hereinafter referred to as GS), dihydropteroate synthase (EC2.5.1.15: hereinafter referred to as DHP), and the like. As thecompounds inhibiting lipid biosynthesis, for example, there arecompounds inhibiting acetyl CoA carboxylase (EC 6.4.1.2: hereinafterreferred to as ACC), and the like. As the compounds inhibiting cell wallbiosynthesis, for example, there are compounds inhibiting cellulosebiosynthesis, and the like. As the compounds influencing proteinbiosynthesis, nucleic acid biosynthesis or cell division, for example,there are compounds inhibiting formation of microtubules, and the like.

Examples of the compounds having plant growth regulator activitiesinclude compounds having antagonistic activities against plant hormoneswhich enhance cell elongation and differentiation, and the like.Specifically, for example, there are 2,4-D, phenoxyalkane carboxylicacid, derivatives of benzoic acid, derivatives of picolinic acid, andthe like.

As the above-described PPO inhibitory-type herbicidal compounds, forexample, there are the compounds disclosed in Duke, S. O., Rebeiz, C.A., ACS Symposium Series 559, Porphyric Pesticides, Chemistry,Toxicology, and Pharmaceutical Applications, American Chemical Society,Washington D.C. (1994), and the like. Specifically, examples thereofinclude the following compounds:

(1) chlormethoxynil, bifenox, chlornitrofen (CNP), acifluorfen(5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitorobenzoic acid) and itsethyl ester, acifluorfen-sodium, oxyfluorfen(2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-trifluorobenzene), oxadiazon(3-[2,4-dichloro-5-(1-methylethoxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-oxydiazol-2-(3H)-one),2-[4-chloro-2-fluoro-5-(prop-2-ynyloxy)phenyl]-2,3,4.5,6,7-hexahydro-1H-isoindol-1,3-dione,chlorphthalim, (N-(4-chlorophenyl)-3,4,5,6-tetrahydrophtalimide),TNPP-ethyl (ethyl2-[1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-5-oxy]propionate), orN3-(1-phenylethyl)-2,6-dimethyl-5-propyonylnicotinamide;

(2) a compound represented by the general formula: J-G (I), wherein G isa group represented by any one of the following general formulas G-1 toG-9 and J is a group represented by any one of the following generalformulas J-1 to J-30:

wherein the dotted lines in the formulas J-5, J-6, J-12 and J-24represent that the left hand ring contains only single bonds, or onebond in the ring is a double bond between carbon atoms;

X is oxygen atom or sulfur atom;

Y is oxygen atom or sulfur atom;

R¹ is hydrogen atom or halogen atom;

R² is hydrogen atom, C₁-C₈alkyl group, C₁-C₈ haloalkyl group, halogenatom, OH group, OR²⁷ group, SH group, S(O)_(p)R²⁷ group, COR²⁷ group,CO₂R²⁷ group. C(O)SR²⁷ group. C(O)NR²⁹V³⁰ group, CHO group, CR²⁷═NOR³⁶group. CH═CR³⁷CO₂R²⁷ group, CH₂CHR³⁷CO₂R²⁷ group, CO₂N═CR³¹R³² group,nitro group, cyano group, NHSO₂R³³ group, NHSO₂NHR³³ group, NR²⁷R³⁸group, NH₂ group or phenyl group optionally substituted with one or moreand the same or different C₁-C₄ alkyl groups;

p is 0, 1 or 2;

R³ is C₁-C₂ alkyl group, C₁-C₂ haloalkyl group, OCH₃ group, SCH₃ group,OCHF₂ group, halogen atom, cyano group or nitro group;

R⁴ is hydrogen atom, C₁-C₃ alkyl group, C₁-C₃ haloalkyl group or halogenatom;

R⁵ is hydrogen atom, C₁-C₃ alkyl group, halogen atom, C₁-C₃ haloalkylgroup, cyclopropyl group, vinyl group, C₂ alkynyl group, cyano group,C(O)R³⁸ group, CO₂R³⁸ group, C(O)NR³⁸R³⁹ group, CR³⁴R³⁵CN group,CR³⁴R³⁵C(O)R³⁸ group, CR³⁴R³⁵CO₂R³⁸ group, CR³⁴R³⁵C(O)NR³⁸R³⁹ group,CHR³⁴OH group, CHR³⁴OC(O)R³⁸ group or OCHR³⁴OC(O)NR³⁸R³⁹ group, or, whenG is G-2 or G-6, R⁴ and R⁵ may form C═O group together with the carbonatom to which they are attached;

R⁶ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₂-C₆ alkoxyalkyl group,C₃-C₆ alkenyl group or C₃-C₆ alkynyl group;

X¹ is single bond, oxygen atom, sulfur atom, NH group, N(C₁-C₃ alkyl)group, N(C₁-C₃ haloalkyl) group or N(allyl) group;

R⁷ is hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, halogenatom, S(O)₂(C₁-C₆alkyl) group or C(═O)R⁴⁰ group;

R⁸ is hydrogen atom, C₁-C₈ alkyl group. C₃-C₈ cycloalkyl group, C₃-C₈alkenyl group, C₃-C₈ alkynyl group, C₁-C₈ haloalkyl group, C₂-C₈alkoxyalkyl group, C₃-C₈ alkoxyalkoxyalkyl group, C₃-C₈ haloalkynylgroup, C₃-C₈ haloalkenyl group, C₁-C₈ alkylsulfonyl group, C₁-C₈haloalkylsulfonyl group, C₃-C₈ alkoxycarbonylalkyl group, S(O)₂NH(C₁-C₈alkyl) group, C(O)R⁴¹ group or benzyl group whose phenyl ring may besubstituted with R⁴²;

n and m are independently 0, 1, 2 or 3 and m+n is 2 or 3;

Z is CR⁹R¹⁰ group, oxygen atom, sulfur atom, S(O) group, S(O)₂ group orN(C₁-C₄ alkyl) group;

each R⁹ is independently hydrogen atom, C₁-C₃ alkyl group, halogen atom,hydroxyl group, C₁-C₆ alkoxy group, C₁-C₆ haloalkyl group, C₁-C₆haloalkoxy group, C₂-C₆ alkylcarbonyloxy group or C₂-C₆haloalkylcarbonyloxy group;

each R¹⁰ is independently hydrogen atom, C₁-C₃ alkyl group, hydroxylgroup or halogen atom;

R¹¹ and R¹² are independently hydrogen atom, halogen atom, C₁-C₆ alkylgroup, C₃-C₆ alkenyl group or C₁-C₆ haloalkyl group;

R¹³ is hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₃-C₆alkenyl group, C₁-C₆ haloalkenyl group, C₃-C₆ alkynyl group. C₃-C₆haloalkynyl group, HC(═O) group, (C₁-C₄ alkyl)C(═O) group or NH₂ group;

R¹⁴ is C₁-C₆ alkyl group, C₁-C₆ alkylthio group, C₁-C₆ haloalkyl groupor N(CH₃)₂ group;

W is nitrogen atom or CR¹⁵;

R¹⁵ is hydrogen atom, C₁-C₆ alkyl group, halogen atom, or phenyl groupoptionally substituted with C₁-C₆ alkyl group, one or two halogen atoms,C₁-C₆ alkoxy group or CF₃ group;

each Q is independently oxygen atom or sulfur atom;

Q¹ is oxygen atom or sulfur atom;

Z¹ is CR¹⁶R¹⁷ group, oxygen atom, sulfur atom, S(O) group, S(O)₂ groupor N(C₁-C₄alkyl) group;

each R¹⁶ is independently hydrogen atom, halogen atom, hydroxyl group,C₁-C₆ alkoxy group, C₁-C₆ haloalkyl group, C₁-C₆ haloalkoxy group, C₂-C₆alkylcarbonyloxy group or C₂-C₆ haloalkylcarbonyloxy group;

each R¹⁷ is independently hydrogen atom, hydroxyl group or halogen atom;

R¹⁸ is C₁-C₆ alkyl group, halogen atom or C₁-C₆ haloalkyl group;

R¹⁹ and R²⁰ are independently hydrogen atom, C₁-C₆ alkyl group, or C₁-C₆haloalkyl group;

Z² is oxygen atom, sulfur atom, NR⁹ group or CR⁹R¹⁰ group;

R²¹ and R²² are independently C₁-C₆ alkyl group, C₁-C₆ haloalkyl group,C₃-C₆ alkenyl group, C₃-C₆ haloalkenyl group, C₃-C₆ alkynyl group orC₃-C₆ haloalkynyl group;

R²³ is hydrogen atom, halogen atom or cyano group;

R²⁴ is C₁-C₆ alkylsulfonyl group, C₁-C₆ alkyl group, C₁-C₆ haloalkylgroup, C₃-C₆ alkenyl group, C₃-C₆ alkynyl group, C₁-C₆ alkoxy group,C₁-C₆ haloalkoxy group or halogen atom;

R²⁵ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₃-C₆ alkenyl group orC₃-C₆ alkynyl group;

R²⁶ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group or phenyl groupoptionally substituted with C₁-C₆ alkyl, one or two halogen atoms, oneor two nitro groups, C₁-C₆ alkoxy group or CF₃ group;

W¹ is nitrogen atom or CH group;

T is a group represented by any one of the following general formulasT-1, T-2 and T-3;

(wherein E¹, E², E³, E⁴, E⁵, E⁶, E⁷, E⁸, E⁹, E¹⁰, E¹¹ and E¹² areindependently hydrogen atom or C₁-C₃ alkyl group);

R²⁷ is C₁-C₈ alkyl group, C₃-C₈ cycloalkyl group. C₃-C₈ alkenyl group,C₃-C₈alkynyl group, C₁-C₈ haloalkyl group, C₂-C₈ alkoxyalkyl group,C₂-C₈ alkylthioalkyl group, C₂-C₈ alkylsulfinylalkyl group, C₂-C₈alkylsulfonylalkyl group, C₁-C₈ alkylsulfonyl group, phenylsulfonylgroup whose phenyl ring may be substituted with at least one substituentselected from the group consisting of halogen atom and C₁-C₄ alkylgroup, C₄-C₈ alkoxyalkoxyalkyl group, C₄-C₈ cycloalkylalkyl group, C₆-C₈cycloalkoxyalkyl group, C₄-C₈ alkenyloxyalkyl group, C₄-C₈alkynyloxyalkyl group, C₃-C₈ haloalkoxyalkyl group, C₄-C₈haloalkenyloxyalkyl group, C₄-C₈ haloalkynyloxyalkyl group, C₆-C₈cycloalkylthioalkyl group, C₄-C₈ alkenylthioalkyl group, C₄-C₈alkynylthioalkyl group, C₁-C₄ alkyl group substituted with phenoxy groupwhose ring is substituted with at least one substituent selected fromthe group consisting of halogen atom, C₁-C₃ alkyl group and C₁-C₃haloalkyl group, benzyloxy group whose ring is substituted with at leastone substituent selected from the group consisting of halogen atom,C₁-C₃ alkyl group and C₁-C₃ haloalkyl group, C₄-C₈ trialkylsilylalkylgroup, C₃-C₈ cyanoalkyl group, C₃-C₈ halocycloalkyl group, C₃-C₈haloalkenyl group, C₃-C₈ alkoxyalkenyl group, C₅-C₈ haloalkoxyalkenylgroup, C₅-C₈ alkylthioalkenyl group, C₃-C₈ haloalkynyl group, C₅-C₈alkoxyalkynyl group, C₅-C₈ haloalkoxyalkynyl group, C₅-C₈alkylthioalkynyl group. C₂-C₈ alkylcarbonyl group, benzyl group whosering is substituted with a! least one substituent selected from thegroup consisting of halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkylgroup, CHR³⁴COR²⁸ group, CHR³⁴COOR²⁸ group, CHR³⁴P(O)(OR²⁸)₂ group,CHR³⁴P(S)(OR²⁸)₂ group, CHR³⁴C(O)NR²⁹R³⁰ group or CHR³⁴C(O)NH₂ group;

R²⁸ is C₁C₆ alkyl group, C₂-C₆ alkenyl group, C₃-C₆ alkynyl group ortetrahydrofuranyl group;

R²⁹ and R³¹ are independently hydrogen atom or C₁-C₄ alkyl group;

R³⁰ and R³² are independently C₁-C₄ alkyl group or phenyl group whosering may be substituted with at least one substituent selected from thegroup consisting of halogen atom, C₁-C₃ alkyl group and C₁-C₃ haloalkylgroup; or,

R²⁹ and R³⁰ together may form —(CH₂)₅—, —(CH₂)₄— or —CH₂CH₂OCH₂CH₂—, orthe ring thus formed may be substituted with at least one substituentselected from the group consisting of C₁-C₃ alkyl group, phenyl groupand benzyl group; or,

R³¹ and R³² may from C₃-C₈ cycloalkyl group together with the carbonatom to which they are attached;

R³³ is C₁-C₁ alkyl group, C₁-C₄ haloalkyl group or C₃-C₆ alkenyl group;

R³⁴ and R³⁵ are independently hydrogen atom or C₁-C₄ alkyl group;

R³⁶ is hydrogen atom, C₁-C₆ alkyl group, C₃-C₆ alkenyl group or C₃-C₆alkynyl group;

R³⁷ is hydrogen atom, C₁-C₄ alkyl group or halogen atom;

R³⁸ is hydrogen atom, C₁-C₆ alkyl group, C₃-C₆ cycloalkyl group, C₃-C₆alkenyl group, C₃-C₆ alkynyl group, C₂-C₆ alkoxyalkyl group, C₁-C₆haloalkyl group, phenyl group whose ring may be substituted with atleast one substituent selected from the group consisting of halogenatom, C₁-C₄ alkyl group and C₁-C₄ alkoxy group, —CH₂CO₂(C₁-C₄ alkyl)group or —CH(CH₃)CO₂(C₁-C₄ alkyl) group;

R³⁹ is hydrogen atom, C₁-C₂ alkyl group or C(O)O(C₁-C₄ alkyl) group;

R⁴⁰ is hydrogen atom, C₁-C₆ alkyl group, C₁-C₆ alkoxy group or NH(C₁-C₆alkyl) group;

R⁴¹ is C₁-C₆ alkyl group, C₁-C₆ haloalkyl group, C₁-C₆ alkoxy group,NH(C₁-C₆ alkyl) group, phenyl group whose ring may be substituted withone substituent selected from the group consisting of R⁴² group, benzylgroup and C₂-C₈ dialkylamino group; and

R⁴² is C₁-C₆ alkyl group, one or two halogen atoms, C₁-C₆ alkoxy groupor CF₃ group;

(3) a compound of the formula (II):

or nipilacrofen,

wherein R⁴³ is C₁-C₄ alkyl group;

R⁴⁴ is C₁-C₄ alkyl group, C₁-C₄ alkylthio group, C₁-C₄ alkoxy group,C₁-C₄ haloalkyl group, C₁-C₄ haloalkylthio group or C₁-C₄ haloalkoxygroup;

R⁴³ and R⁴⁴ together may form —(CH₂)₃— or —(CH₂)₄—;

R⁴⁵ is hydrogen atom or halogen atom;

R⁴⁶ is hydrogen atom or C₁-C₄ alkyl group;

R⁴⁷ is hydrogen atom, nitro group, cyano group, —COOR⁴⁹ group,—C(═X)NR⁵⁰R⁵¹ group or —C(═X²)R³² group;

R⁴⁸ is hydrogen atom, halogen atom, cyano group, C₁-C₄ alkyl groupoptionally substituted with at least one substituent selected from thegroup consisting of halogen atom and hydroxyl group, C₁-C₄ alkoxy group,phenyl group optionally substituted with at least one substituentselected from the group consisting of halogen atom, nitro group, cyanogroup, C₁-C₄alkyl group, C₁-C₄ alkoxy group and halo-C₁-C₄ alkyl group,pyrrolyl group, C₂-C₈ alkyl group, C₃-C₈ alkenyl group, C₃-C₈ alkynylgroup, C₃-C₈ alkoxy group, a group selected from the group consisting ofC₂-C₈ alkyl group, C₃-C₈ alkenyl group, C₃-C₈ alkynyl group and C₃-C₈alkoxy group into which at least one oxygen atom is inserted, or any oneof groups represented by the following formulas:

wherein R⁴⁹, R⁵⁰ and R⁵² are, the same or different, hydrogen atom orC₁-C₄ alkyl group;

R⁵⁰ and R⁵¹ may form saturated alicyclic 5 or 6 membered ring togetherwith the nitrogen atom to which they are attached;

R⁵² is hydrogen atom, C₁-C₄ alkyl group or C₁-C₄ alkyl group substitutedwith at least one halogen atom;

R³³ is hydrogen atom, C₁-C₄ alkyl group optionally substituted with atleast one halogen atom, C₂-C₆ alkenyl group optionally substituted withat least one halogen atom, C₃-C₆ alkynyl group optionally substitutedwith at least one halogen atom, phenyl group optionally substituted withat least one halogen atom, C₃-C₈ cycloalkyl group, cyanomethyl group, orR⁶³CO— group;

R⁵⁴ is hydrogen atom, C₁-C₆ alkyl group optionally substituted with atleast one halogen atom, C₂-C₆ alkenyl group optionally substituted withat least one halogen atom, C₃-C₆ alkynyl group optionally substitutedwith at least one halogen atom, phenyl group optionally substituted withhalogen atom, C₃-C₈ cycloalkyl group, cyanomethyl group, C₁-C₄alkoxy-C₁-C₆ alkyl group, di-C₁-C₄ alkylamino-C₁-C₄ alkyl group,tetrahydrofurfurylmethyl group, C₃-C₆ alkynyloxy-C₁-C₄ alkyl group,benzyl whose ring may be substituted with substituent selected from thegroup consisting of halogen atom, nitro group, cyano group, C₁-C₄ alkylgroup, C₁-C₄ alkoxy group and halo-C₁-C₄ alkyl group, —C(═X²)R⁶³ group,—(CH₂)_(a)—(O)_(d)—R⁷⁰ group, —(CH₂)_(a)—O—(CH₂)_(b)—R⁷⁰ group,—(CH₂)_(a)—X²—R⁷⁶ group;

R⁵³ and R⁵⁴ together with the nitrogen atom to which they are attachedmay form saturated alicyclic 3, 5 or 6 membered ring or aromatic 5 or 6membered ring in which a carbon atom may be optionally replaced withoxygen atom;

R⁵⁵ is hydrogen atom, C₁-C₄ alkyl group, C₂-C₆ alkenyl group or C₃-C₆alkynyl group, or R⁵⁵ and R⁵⁶ together may form —(CH₂)_(e)—;

R⁵⁶ and R⁵⁷ are independently C₁-C₄ alkyl group optionally substitutedwith at least one halogen atom, C₂-C₆ alkenyl group optionallysubstituted with at least one halogen atom, C₃-C₆ alkynyl optionallysubstituted with at least one halogen atom or phenyl group optionallysubstituted with at least one halogen atom, hydrogen atom, C₃-C₆cycloalkyl group, —XR⁶⁰ group or —NR⁶¹R⁶² group;

R⁵⁸ is hydrogen atom, C₁-C₆ alkyl group, C₂-C₆ alkenyl group, C₃-C₆alkynyl group, C₁-C₄ alkylcarbonyl group, cyano-C₁-C₃ alkyl group, C₁-C₄alkoxycarbonyl-C₁-C₄ alkyl group, di-C₁-C₄ alkoxycarbonyl-C₁-C₄ alkylgroup, benzyl group, C₁-C₄ alkoxy-C₁-C₄ alkynyl group, —(CH₂)_(a)—R⁷⁵group, —(CH₂)_(a)—X²—R⁷² group, —(CH₂)_(a)—X²—(CH₂)_(b)—R⁷² group or—(CH₂)_(a)—X²—(CH₂)_(b)—X²—(CH₂)_(c)—R⁷² group;

R⁵⁹ is hydrogen atom, C₁-C₄ alkyl group, C₂-C₆ alkenyl group, C₃-C₆alkynyl group, cyano-C₁-C₃ alkyl group. C₁-C₄ alkylcarbonyl-C₁-C₃ alkylgroup or phenyl group;

R⁶⁰ is C₁-C₄ alkyl group optionally substituted with at least onehalogen atom;

R⁶¹ and R⁶² are, the same or different, hydrogen atom or C₁-C₄ alkylgroup;

R⁶³ is C₁-C₄ alkyl group optionally substituted with at least onehalogen atom, C₁-C₄ alkoxy-C₁-C₄ alkyl group. C₁-C₄ alkylthio-C₁-C₄alkyl group, C₃-C₆ cycloalkyl group, phenyl group whose ring may besubstituted with one substituent selected from the group consisting ofhalogen atom, nitro group, cyano group, C₁-C₄ alkyl group, C₁-C₄ alkoxygroup and halo-C₁-C₄ alkyl group, —NR⁷³R⁷⁴ group or—(CH₂)_(n)—(O)_(d)—R⁷⁵ group;

R⁶⁴ is C₁-C₄ alkoxycarbonyl group or carboxyl group;

R⁶⁵ is chloromethyl group, cyanomethyl group, C₃-C₆ cycloalkyl groupinto which at least one oxygen atom may be inserted, or C₁-C₄alkoxycarbonyl-C₁-C₄ alkyl group;

R⁶⁶ is hydroxyl group or —NR⁶⁷R⁶⁸ group;

A is —NR⁶⁷R⁶⁸ group or —S(O)_(i)—R⁶⁹ group;

R⁶⁷ and R⁶⁸ are, the same or different, hydrogen atom or C₁-C₄ alkylgroup;

R⁶⁹ is C₁-C₁ alkyl group or C₁-C₄ haloalkyl group;

R⁷⁰ is hydrogen atom, hydroxyl group, halogen atom, C₁-C₄ alkyl groupoptionally substituted with at least one C₁-C₄ alkoxy group, C₃-C₆cycloalkyl group into which at least one oxygen atom may be inserted,C₃-C₆ cycloalkyl group optionally substituted with one or two methylgroups, furyl group, thienyl group or —C(═O)R⁷¹ group;

R⁷¹ and R⁷² are, the same or different, C₁-C₄ alkyl group or C₁-C₄alkoxy group;

R⁷³ and R⁷⁴ are, the same or different, C₁-C₄ alkyl group or phenylgroup;

R⁷⁵ is C₃-C₆ cycloalkyl into which at least one oxygen atom may beinserted, C₃-C₆ cycloalkyl group optionally substituted with one or twomethyl groups, furyl group, thienyl group or —C(═O)R⁷¹ group;

R⁷⁶ is C₁-C₄ alkyl group;

a, b and c is independently 1, 2 or 3;

d is 0 or 1;

e is 2 or 3

f is 1 or 2; and

X² is oxygen atom or sulfur atom.

In addition, as other N-substituted pyrazoles, there are the3-substituted-2-aryl-4,5,6,7-tetrahydro-indazoles described in Lyga etal., Pesticide Sci., 42: p 29 (1994), and the like.

As specific examples of the compounds inhibiting electron transfer inphotochemical system I, for example, there are paraquat, diquat, and thelike. As specific examples of the compounds inhibiting electron transferin photochemical system II, for example, there are triazine compounds(e.g., atrazine, etc.), urea compounds (e.g., diuron, etc.), nitrilecompounds (e.g., bromoxynil and ioxynil) and the like. As specificexamples of the compounds inhibiting 4-HPPD, for example, there areisoxazoles (e.g., isoxaflutole), pyrazoles, triketones, and the like. Asspecific examples of the compounds inhibiting PDS, for example, thereare norflurazon, flurochloridone, fluridone, flurtamone, diflufenican,and the like. As specific examples of the compounds inhibiting EPSPS,for example, there are glyphosate, and the like. As specific examples ofthe compounds inhibiting ALS, for example, there are sulfonylureas,imidazolinones, pyrimidinylthiobenzoates, triazolopyrimidines, and thelike. As specific examples of the compounds inhibiting GS, for example,there are bialaphos, glufosinate, and the like. As specific examples ofthe compounds inhibiting DHP, for example, there are asulam, and thelike. As specific examples of the compounds inhibiting ACC, for example,there are cyclohexanediones, aryloxyphenoxypropionates, and the like. Asspecific examples of the compounds inhibiting cellulose, for example,there are dichlobenil, and the like.

Various examples of the weed control compounds useful in the presentinvention are shown by the following chemical structures:

In the first aspect of the method of the present invention, the genes tobe used are those encoding proteins having the following characteristics(a) to (c) (hereinafter sometimes referred to as the objectiveproteins):

(a) having a specific affinity for weed control substances;

(b) having substantially no capability of modifying substances for whichsaid protein has a specific affinity; and

(c) being substantially free from framework regions of variable regionsof an immunoglobulin.

The term “a specific affinity” for weed control substances of the abovecharacteristic (a) means that an enzyme (the objective protein) and asubstrate (the weed control substance), or an enzyme (the objectiveprotein) and an inhibitor or a regulator of an activity of the enzyme(the weed control substance) bind to each other, enzymatically; or thatthe objective protein and the weed control substance bind to each otheron the basis of affinity and specificity, such as those shown in areceptor-chemical bond, for example, a bond between a receptor and aligand, and the like. The objective proteins may be naturally occurringproteins; variants thereof obtained by introduction of amino acidsubstitution, addition, deletion, modification and the like intonaturally occurring proteins; and artificially synthesized proteinshaving random amino acid sequences selected with the guidance of theiraffinity for weed control substances, in so far as they have structuresspecifically binding to weed control substances.

The term “having substantially no capability of modifying” in thecharacteristic (b) means that enzymatic reactivity with substances forwhich said protein has a specific affinity is substantially inactive ornot existed (except the specific affinity for weed control substances inthe characteristic (a)). Examples of this include a case that theobjective protein does not have any capability of converting a substancefor which said protein has a specific affinity such as a certain weedcontrol substance or a substance having an essential part of thestructure of the substrates on the basis of a specific affinity for saidprotein, and the like to a substance having a chemical structuredifferent from that of the substance for which said protein has aspecific affinity. The protein “having substantially no capability ofmodifying” can be, for example, identified by checking non-recovery ofthe growth of a microorganism whose gene encoding the said protein isdeleted and thus cannot grow under a usual condition in a case where thegene encoding the said protein is introduced into the microorganism insuch a state that the introduced gene is expressed in the microorganism.

The term “substantially free from the framework regions of variableregions of an immunoglobulin” in the characteristic (c) mean that theobjective protein does not form a stereostructure specific for thevariable regions of an immunoglobulin. The term “framework regions ofvariable regions of an immunoglobulin” mean regions remaining afterremoving the hypervariable regions from the variable regions of H chainand L chain which are the constituents of the immunoglobulin molecule.In these regions, conservation of the amino acid sequences is relativelyhigh and these regions function for maintaining the highly conservedstereostructure of the variable regions. Due to formation of the abovestereostructure, the hypervariable regions separately located at threesites on respective H chain and L chain are collected to one site on thestereostructure to form an antigen binding site [Alberts, B., et al. ed.(1983), Molecular Biology of the Cell, p 979, Garland Publishing. Inc.,New York].

The objective protein having the above characteristic (c) can beselected on the basis of, for example, the amino acid sequences of theproteins. As specific examples of the protein, there are a protein whichdoes not contain any amino acid sequence composed of about 30 aminoacids or more and having about 60% or more homology with the known aminoacid sequences of the framework regions of the variable regions of animmunoglobulin, and the like. For example, the presence or absence ofthe above framework regions can be confirmed by PCR using a geneencoding the protein as a template and DNAs having nucleotide sequencesencoding the variable regions derived from H chain or L chain of theimmunoglobulin as amplification primers, for example, the primers VH1BACK and VH1FOR-2, or VK2BACK and VK4FOR described by Clackson, T. etal., Nature 352; p 624 (1991), or primers contained in a commerciallyavailable kit for cloning recombinant antibody genes, for example, Heavyprimer mix or Light primer mix of Recombinant Phage Antibody System(Pharmacia Biotech) to analyze presence or absence of amplification ofDNA having a given length. Examples of the binding proteins having aspecific affinity for weed control substances also include peptideshaving an affinity for the weed control substances.

Specific examples of the objective proteins having the abovecharacteristics of (a) to (c) include inactive-type binding proteinshaving an affinity for protoporphyrin IX [e.g., inactive-type magnesiumchelatase whose substrate is protoporphyrin IX (the weed controlsubstance), inactive-type ferrochelatase (protoheme ferrolyase; EC4.9.9.1), inactive-type cobalt chelatase which catalyzes a chelatingreaction of a cobalt ion with a compound having tetrapyrrole ring as asubstrate, peptides having an affinity for protoporphyrin IX, i.e.,proteins composed of 4 to 100 amino acids (for example, peptide HASYShaving an affinity for protoporphyrin IX, e.g., a protein comprising theamino acid sequence of SEQ ID NO: 53 and a protein having the amino acidsequence of SEQ ID NO: 54; peptide RASSL having an affinity forprotoporphyrin IX, e.g., a protein comprising the amino acid sequence ofSEQ ID NO: 55 and a protein having the amino acid sequence of SEQ ID NO:56; peptide YAGY having an affinity for porphyrin compounds, e.g., aprotein comprising the amino acid sequence of SEQ ID NO: 57 and aprotein having the amino acid sequence of SEQ ID NO: 58; peptide YAGFhaving affinity for porphyrin compounds, e.g., a protein comprising theamino acid sequence of SEQ ID NO: 59 and a protein having the amino acidsequence of SEQ ID NO: 60; and the like)], inactive-type bindingproteins having an affinity for protoporphyrinogen IX (e.g.,inactive-type PPO, inactive-type coproporphyrinogen III oxidase), andthe like.

The above inactive-type binding proteins include variants thereof whoseactivities have been lost by amino acid substitution, addition,deletion, modification and the like of naturally occurring activeproteins under natural or artificial conditions.

Cellular dysfunction caused by weed control substances can be preventedby binding of these binding, proteins to the weed control substances inplant cells to exhibit the desired weed control compound-resistance.

The inactive-type magnesium chelatase is protoporphyrin IX bindingsubunit protein of magnesium chelatase, or its variant having a specificaffinity for protoporphyrin IX, and specific examples thereof includethe subunit protein from which its organelle transit signal sequence hasbeen deleted, and the like.

The inactive-type ferrochelatase is its variant having no capability ofmodifying protoporphyrin IX and having a specific affinity forprotoporphyrin IX, and specific examples thereof include aferrochelatase variant in which a region presumed to be a Fe ion bindingsite of ferrochelatse has been modified, and the like.

The inactive-type cobalt chelatase is a substrate binding subunitprotein of cobalt chelatase, or its variant having no capability ofmodifying protoporphyrin IX and having a specific affinity forprotoporphyrin IX.

The inactive-type PPO is its variant having no capability of oxidizingprotoporphyrinogen IX and having a specific affinity forprotoporphyrinogen IX, and specific examples thereof include a PPOvariant in which a region presumed to be FAD binding site of PPO (aregion having the amino acid sequence GXGXXG (SEQ ID NO: 77) wherein Xis any amino acid, e.g., a region comprising the 63rd to 68th aminoacids from the N-terminus of chloroplast localized PPO of mouse-earcress (Arabidopsis thaliana) and having the amino acid sequence ofGGGISG (SEQ ID NO: 78) has been deleted, and the like. The inactive-typecoproporphyrinogen III oxidase is its variant having no capability ofoxidizing protoporphyrinogen IX and having a specific affinity forprotoporphyrinogen IX.

The genes encoding the above proteins can be obtained by, for example,as follows.

The genes encoding protoporphyrin IX binding subunit protein ofmagnesium chelatase, for example, those derived from the photosyntheticbacterium, Rhodobacter capsulatus (Genebank accession M74001), mouse-earcress (Genebank accession Z68495), barley (Genebank accession U96216),snapdragon (Antirrhinum majus) (Genebank accession U26916),Synechocystis P.C.C. 6803 (Genebank accession U29131) and the like havebeen known. For isolating such a known gene (its nucleotide sequence hasbeen known), PCR can be carried out by using genomic DNA or cDNA of anorganism having the desired gene as a template and primers produced onthe basis of nucleotide sequences corresponding to those about the N-and C-termini of the protein encoded by the gene to amplify the desiredgene. Further, genes encoding protoporphyrin IX binding subunit proteinof magnesium chelatase can be obtained from photosynthetic organismsother than the above. For example, first, a cDNA library is constructedby obtaining mRNA from the desired photosynthetic organism, synthesizingcDNA by using the mRNA as a template with a reverse transcriptase, andintegrating the cDNA into a phage vector such as ZAPII, etc., or aplasmid vector such as pUC, etc. For amplifying a DNA fragmentcontaining at least a part of the gene encoding protoporphyrin IXbinding subunit protein of magnesium chelatase, PCR can be carried outby using the above-constructed cDNA library as a template and primersdesigned and synthesized on the basis of nucleotide sequences wellconserved among known genes such as the above-described genes. Screeningof the cDNA library can be carried out by using the DNA fragment thusobtained as a probe to select positive clones. The desired gene ofprotoporphyrin IX binding subunit protein of magnesium chelatase can beconfirmed by sequence determination of the nucleotide sequence of theselected clone.

For obtaining the gene encoding a variant of protoporhyrin IX bindingsubunit protein of magnesium chelatase having an specific affinity forprotoporphyrin IX, for example, the gene encoding the subunit protein ismutagenized by introduction of nucleotide substitution, addition,deletion, modification and the like, followed by introducing theresultant gene into Escherichia coli BL21(DE3) strain according to themethod described by Gibson, L. C. D. et al., Proc. Natl. Acad. Sci. USA,92; p 1941 (1995) and the like to obtain transformants, and culturingthe transformants under conditions that high expression of the gene thusintroduced occurs. The desired gene encoding a variant of the subunitprotein having a specific affinity for protoporphyrin IX can be obtainedby selecting a strain whose cultured cells have turned red and have thefluorescence absorption showing accumulation of protoporphyrin IX(excitation wavelength 405 nm, emission wavelength 630 nm).

As the genes encoding ferrochelatase, for example, those derived fromEscherichia coli (Genebank accession D90259), Bacillus subtilis(Genebank accession M97208), Bradyrhizobium japonicum (Genebankaccession M92427), yeast Saccharomyces cerevisiae (Genebank accessionJ05395), mouse (Genebank accession J05697), human being (Genebankaccession D00726), barley (Genebank accession D26105), cucumber(Genebank accession D26106), and the like have been known. For isolatingsuch a known gene (its nucleotide sequence has been known), PGR can becarried out by using genomic DNA or cDNA of an organism having thedesired gene as a template and primers produced on the basis ofnucleotide sequences corresponding to those about the N- and C-terminiof the protein encoded by the gene to amplify the desired gene. Further,for obtaining other genes encoding ferrochelatase, for example, first, acDNA library is constructed by obtaining mRNA from the desired organism,synthesizing cDNA by using the mRNA as a template with a reversetranscriptase, and integrating the cDNA into a phage vector such asZAPII, etc. or a plasmid vector such as pUC, etc. The cDNA library canbe introduced into ferrochelatase deficient mutant strain of Escherichiacoli VS200 described by Miyamoto, K, et al., Plant Physiol., 105; p 769(1994), followed b subjecting a complementation test to select clonescontaining ferrochelatase gene derived from the desired organism.Further, for amplifying a DNA fragment, PCR can be carried out by usingthe above-constructed cDNA library as a template and primers prepared onthe basis of nucleotide sequences well conserved among known genes suchas the above-described genes. Screening of the cDNA library can becarried out by using the DNA fragment thus obtained as a probe to selectpositive clones. The desired ferrochelatase gene can be confirmed bysequence determination of the nucleotide sequence of the selected clone.

For obtaining the gene encoding a variant of ferrochelatase having nocapability of modifying protoporphyrin IX and having a specific affinityfor protoporphyrin IX (for example, the gene encoding a ferrochelatasevariant in which the region presumed to be a Fe ion binding site offerrochelatase is modified), PCR can be carried out by preparing amutagenesis primer for introduction of mutation into the region on thebasis of nucleotide sequence encoding the amino acid sequence about theregion, and using a commercially available site-directed mutagenesis kit(Mutan-Super Express, Takara Shuzo) to obtain the gene encoding theabove variant. Specifically, a wild type ferrochelatase gene is insertedinto the cloning site of plasmid vector pKF19K and PCR is carried out byusing the resultant plasmid DNA as a template, the above-describedmutagenesis primer and a selection primer for restoration of ambermutation located on kanamycin resistant gene of pKF19K. The geneamplified by PCR is introduced into Escherichia coli MV1184 (suppressorfree strain) and the transformants are screened according to kanamycinresistance to isolate Escherichia coli having ferrochelatase gene inwhich the nucleotide sequence corresponding to the amino acid sequencewhich constitutes the desired region has been modified. The isolatedgene can be confirmed as the gene encoding the desired protein byanalyzing the nucleotide sequence of the plasmid DNA of the Escherichiacoli.

The genes encoding the peptides having an affinity for protoporphyrinIX, i.e., the proteins composed of 4 to 100 amino acids can be obtainedby synthesizing a peptide library according to, for example, thecombinatorial chemistry method as described by Sugimoto, N., Nakano, S.,Chem., Lett., p 939 (1997) and the like, selecting a peptide having anaffinity for the weed control substance, analyzing the amino acidsequence of the peptide thus selected with a peptide sequencer,designing a gene containing a nucleotide sequence encoding the aminoacid sequence, and synthesizing the nucleotide sequence with a DNAsynthesizer or the like.

Further, a phase clone displaying a peptide having an affinity for theweed control substance can be selected from a phage library according tophage display method. Specifically, for example, a phage librarydisplaying a protein having a random amino acid sequence on the surfaceof M13 phage particles is constructed by inserting a nucleotide sequenceencoding the protein having the random amino acid sequence into theupstream from the region encoding the coat protein pill of M13 phagegene. On the other hand, the weed control substance labeled with biotinis bound to a plate coated with avidin or streptoavidin to prepare asupport coated with the weed control substance. A phage displaying thedesired protein having an affinity for the weed control substance can beisolated by screening the above phage library on the plate coated withthe weed control substance and the gene of the desired protein can beobtained from the isolated phage.

The gene encoding a protein containing the repetition of the amino acidsequence represented by SEQ ID NO: 53, 55, 57 or 59 four times or eighttimes can be produced by, for example, selecting a nucleotide sequencein which the nucleotide sequence encoding the above amino acid sequenceis repeated the given times after the initiation codon ATG, synthesizingan oligonucleotide comprising the selected nucleotide sequence and anoligonucleotide comprising a nucleotide sequence complementary to theselected nucleotide sequence by a DNA synthesizer, and then subjectingthem to annealing. Further, the genes encoding the amino acid sequencerepresented by SEQ ID NO: 54, 56, 58 or 60 can be produced by selectinga nucleotide sequence encoding the amino acid sequence, synthesizing anoligonucleotide comprising the selected nucleotide sequence and anotheroligonucleotide comprising a nucleotide sequence complementary to theselected nucleotide sequence by a DNA synthesizer, and then subjectingthem to annealing. In this respect, for selecting the nucleotidesequence encoding the given amino acid sequence, for example, it ispreferred to select codons frequently used in genes derived from plants.

As PPO genes, for example, those derived from Escherichia coli (Genebankaccession X68660), Bacillus subtilis (Genebank accession M97208),Haemophilus influenzae (Genebank accession L42023), mouse (Genebankaccession D45185), human being (Genebank accession D38537), mouse-earcress (Genebank accession D83139), tobacco (Genebank accession Y13465,Y13466) and the like have been known. For isolating such a known gene(its nucleotide sequence has been known), PCR is carried out by usinggenomic DNA or cDNA of an organism having the desired gene as a templateand primers produced on the basis of nucleotide sequences correspondingto those about the N- and C-termini of the protein encoded by the geneto amplify the desired gene. Further, for obtaining other PPO genes, forexample, first, a cDNA library is constructed from an organism havingthe desired gene according to the above-described method. The cDNAlibrary can be introduced into Escherichia coli PPO deficient mutantstrain VSR800 described by Narita, S., et al., Gene, 182; p 169 (1996),followed by subjecting a complementation test to select clonescontaining PPO gene derived from the desired organism. Further, foramplifying a DNA fragment, PCR can be carried out by using theabove-constructed cDNA library as a template and primers prepared on thebasis of nucleotide sequences well conserved among known genes such asthe above-described genes. Screening of the cDNA library can be carriedout by using the DNA fragment thus obtained as a probe to selectpositive clones. The desired PPO gene can be confirmed by sequencedetermination of the nucleotide sequence of the selected clone.

For obtaining the gene encoding a variant of PPO having no capability ofoxidizing protoporphyrinogen IX and having a specific affinity forprotoporphyrinogen IX, for example, PPO gene is mutagenized byintroducing nucleotide substitution, addition, deletion, modification,etc. and the resultant modified gene is introduced into the aboveEscherichia coli whose growth is inhibited light-dependently bytreatment with a PPO inhibitory-type herbicidal compound. A geneencoding a protein having protoporphyrinogen IX binding capability canbe selected by culturing the Escherichia coli thus obtained in thepresence of hemin, aminolevulinic acid and a PPO inhibitory-typeherbicidal compound to select a clone which can grow even in the light.A gene encoding a protein having no capability of oxidizingprotoporphyrinogen IX can be selected by expressing the modified genethus selected in a host such as Escherichia coli, etc. to prepare aprotein encoded by the gene, and measuring its capability of oxidizingprotoporphyrinogen IX according to the method described by Jacobs, N. J.and Jacobs, J. M. (1982) Enzyme, 28, 206-219 and the like. Morespecifically, the above modified gene is inserted into an expressionvector for Escherichia coli and introduced into PPO gene (hemG locus)deficient mutant of Escherichia coli such as Escherichia coli BT3 straindescribed by Yamamoto, F., et al., Japanese J. Genet., 63; p 237 (1988)and the like. The Escherichia coli is cultured in a culture mediumcontaining hemin and aminolevulinic acid in addition to the cell growthinhibitor corresponding to the selection marker of the vector introducedinto the Escherichia coli to obtain transformants. The protein encodedby the modified gene can be produced from the transformant. Further, agene which does not complement PPO gene deficiency of its host cell canbe obtained by culturing the transformant in a culture mediumsubstantially free from hemin and aminolevulinic acid to identify astrain which does not grow. This latter method can also be used forselection of the gene encoding a protein having no capability ofoxidizing protoporphyrinogen IX.

Further, for obtaining the gene encoding a variant of PPO in which theregion presumed to be a FAD binding site of PPO (the region having theamino acid sequence GXGXXG, wherein X is any amino acid) is deleted,first, a mutagenesis primer for introduction of deletion mutation of theregion is prepared on the basis of the nucleotide sequence encoding theamino acid sequence about the region. Then, PCR is carried out by usingthe mutagenesis primer and a commercially available site-directedmutagenesis kit (Mutan-Super Express, Takara Shuzo) as described aboveto obtain the gene encoding the above variant protein in which theregion has been deleted.

The genes encoding peptide proteins such as the peptides HASYS and RASSLhaving an affinity for protoporphyrin IX, and the peptides YAGA and YAGFhaving an affinity for prophyrin compounds, and the like can be obtainedby subjecting oligonucleotides synthesized by a DNA synthesizer toannealing.

Furthermore, genes encoding unknown peptide proteins having affinitiesfor other weed control substances can be produced by the followingmethods and the like. For example, various peptide libraries can beconstructed according to, for example, the combinatorial chemistrymethod as described by Sugimoto, N., Nakano, S., Chem., Lett., p 939(1997), and the like. Peptides are selected from the peptide librariesthus constructed with the guidance of affinities for weed controlsubstances, followed by analyzing the amino acid sequences of thepeptides with a peptide sequencer. Thus, genes encoding the peptides canbe synthesized by a DNA synthesizer. Alternatively, phase clonesdisplaying peptides having affinities for weed control substances can beobtained by selecting phage libraries according to phage display method.Specifically, for example, a phage library displaying a protein having arandom amino acid sequence on the surface of M13 phage particles isconstructed by inserting a nucleotide sequence encoding the proteinhaving the random amino acid sequence into the upstream from the regionencoding the coat protein pIII of M13 phage gene. On the other hand, aweed control substance labeled with biotin is bound to a plate coatedwith avidin or streptoavidin to prepare a support coated with the weedcontrol substance. A phage displaying the desired protein having anaffinity for the weed control substance can be isolated by screening theabove phage library on the plate coated with the weed control substanceand the gene of the desired protein can be obtained from the isolatedphage.

As the genes encoding coproporphyrinogen III oxidase, for example, thosederived from Escherichia coli (Genebank accession X75413), Salmonellatyphimurium (Genebank accession L19503), yeast Saccharomyces cerevisiae(Genebank accession J03873), mouse (Genebank accession D1633), humanbeing (Genebank accession D16333), soybean (Genebank accession X71083),barley (Genebank accession X82830), tobacco (Genebank accession X82831)and the like have been known. For isolating such a known gene (itsnucleotide sequence has been known), PCR is carried out by using genomicDNA or cDNA of an organism having the desired gene as a template andprimers produced on the basis of nucleotide sequences corresponding tothose about the N- and C-termini of the protein encoded by the gene toamplify the desired gene. Further, for obtaining othercoproporphyrinogen III oxidase genes, for example, first, a cDNA libraryis constructed from an organism having the desired gene by preparingmRNA from the desired organism, synthesizing cDNA using the mRNA as atemplate with a reverse transcriptase and integrating this into aplasmid vector such as pRS313 described by Sikorski, R. S., et al.,Genetics, 122; p 19 (1989), and the like. The cDNA library can beintroduced into yeast coproporphyrinogen III oxidase deficient mutantstrain HEM13 described by Troup, B., et al., Bacterid., 176; p 673(1994), followed by subjecting a complementation test to select clonescontaining coproporphyrinogen III oxidase derived from the desiredorganism. Further, for amplifying a DNA fragment, PCR can be carried outby using the above-constructed cDNA library as a template and primersprepared on the basis of nucleotide sequences well conserved, amongknown genes such as the above-described genes. Screening of the cDNAlibrary can be carried out by using the DNA fragment thus obtained as aprobe to select positive clones. The desired coproporphyrinogen IIIoxidase gene can be confirmed by sequence determination of thenucleotide sequence of the selected clone.

For obtaining the gene encoding a variant of coporphyrinogen III oxidasehaving no capability of oxidizing protoporphyrinogen IX and having aspecific affinity for protoporphyrinogen IX, for example,coproporphyrinogen III oxidase gene is mutagenized by introducingnucleotide substitution, addition, deletion, modification, etc., and theresultant gene is introduced into the above Escherichia coli whosegrowth is inhibited light-dependently by treatment with a PPOinhibitory-type herbicidal compound. A gene encoding a protein havingprotoporphyrinogen IX binding capability can be selected by culturingthe Escherichia coli thus obtained in the presence of hemin,aminolevulinic acid and a PPO inhibitory-type herbicide to select aclone which can grow even in the light. A gene encoding a protein havingno capability of oxidizing protoporphyrinogen IX can be selected byexpressing the modified gene thus selected in a host such as Escherichiacoli, etc. to prepare a protein encoded by the gene, and measuring itscapability of oxidizing protoporphyrinogen IX according to the methoddescribed by Jacobs, N. J. and Jacobs, J. M. (1982) Enzyme, 28, 206-219and the like.

The genes which is used in the second aspect of the method of thepresent invention are those encoding proteins having the followingcharacteristics (a) to (c):

(a) having a specific affinity for protoporphyrin IX;

(b) having substantially no capability of modifying protoporphyrinogenIX; and

(c) being substantially free from framework regions of variable regionsof immunoglobulins.

The term “a specific affinity” for protoporphyrin IX in thecharacteristic (a) is substantially the same as that in the above firstaspect of the method of the present invention and means that the proteinand protoporphyrin IX bind to each other, enzymatically or the proteinand protoporphyrin IX bind to each other on the basis of affinity andspecificity as those shown in receptor chemical bond such as a bondbetween a receptor and a ligand and the like. The proteins may benaturally occurring proteins; variants thereof in which amino acidsubstitution, addition, deletion, modification and the like areintroduced into naturally occurring proteins; and artificiallysynthesized proteins having random amino acid sequences which areselected with the guidance of an affinity for protoporphyrin IX in sofar as they have structures specifically binding to protoporphyrin IX.

The term “having substantially no capability of modifying”protoporphyrinogen IX in the characteristic (b) means that enzymaticreactivity with protoporphyrinogen IX of the protein is substantiallyinactive or not existed. For example, this means that the protein doesnot have capability of converting protoporphyrinogen IX into a substancehaving a chemical structure different from that of protoporphyrinogenIX.

The term “substantially free from framework regions of variable regionsof immunoglobulins” means the same as that in the above first aspect ofthe method of the present invention and the protein does not form thestereostructure specific for the variable regions in the immunoglobulinas is described hereinabove.

As specific examples of the proteins having the above characteristics(a) to (c), there are active or inactive-type binding proteins having anaffinity for protoporphyrin IX [e.g., active or inactive-type magnesiumchelatase whose substrate is protoporphyrin IX, active or inactive-typeferrochelatase, active or inactive-type cobalt chelatase which catalyzesa chelating reaction of a cobalt ion with a compound having tetrapyrrolering as a substrate, peptides, i.e., proteins composed of 4 to 100 aminoacids, having an affinity for protoporphyrin IX (for example, proteinscontaining at least one peptide selected from peptide HASYS having anaffinity for protoporphyrin IX, e.g., a protein comprising the aminoacid sequence of SEQ ID NO: 53 and a protein having the amino acidsequence of SEQ ID NO: 54; peptide RASSL having an affinity forprotoporphyrin IX, i.e., a protein comprising the amino acid sequence ofSEQ ID NO: 55 and a protein having the amino acid sequence of SEQ ID NO:56: peptide YAGY having an affinity for porphyrin compounds, e.g., aprotein comprising the amino acid sequence of SEQ ID NO: 57 and aprotein having the amino acid sequence of SEQ ID NO: 58; peptide YAGFhaving affinity for porphyrin compounds, i.e., a protein comprising theamino acid sequence of SEQ ID NO: 59 and a protein having the amino acidsequence of SEQ ID NO: 60; and the like)], and the like.

The genes encoding the above proteins can be obtained by, for example,as follows.

Active-type magnesium chelatases are composed of three heterogenoussubunit proteins, i.e., protoporhyrin IX binding subunit protein (Hsubunit protein), I subunit protein and D subunit protein, all of themare essential for catalytic activity. Three independent subunit proteinsare encoded by different genes. The genes of protoporphyrin IX bindingsubunit protein can be obtained by PCR or screening of cDNA library asdescribed hereinabove.

As the gene encoding I subunit protein of a magnesium chelatase, forexample, those derived from photosynthetic bacterium, Rhodobactersphaeroides (Genebank accession AF017642), Rhodobacter capsulatus(Genebank accession Z11165), Arabidopsis (Genebank accession D49426),barley (Genebank accession U26545), soybean (Genebank accession D45857),tobacco (Genebank accession AF14053), Synechocystis P.C.C.6803 (Genebankaccession U35144) and the like have been known. For isolating such aknown gene (its nucleotide sequence has been known), PCR can be carriedout by using genomic DNA or cDNA of an organism having the desired geneas a template and primers produced on the basis of nucleotide sequencescorresponding to those about the N- and C-termini of the protein encodedby the desired gene. Further, genes encoding I subunit protein of amagnesium chelatase can be obtained from photosynthetic organisms otherthan the above. For example, first, a cDNA library is constructed byobtaining mRNA from the desired photosynthetic organisms, synthesizingcDNA by using the mRNA as a template with a reverse transcriptase, andintegrating the cDNA into a phage vector such as ZAPII, etc., or plasmidvector such as pUC, etc. For amplifying a DNA fragment containing atleast a part of the gene encoding I subunit protein of a magnesiumchelatase, PCR can be carried out by using the above-constructed cDNAlibrary as a template and primers designed and synthesized on the basisof nucleotide sequences well conserved among known genes such as theabove described genes. Screening of the cDNA library can be carried outby using the DNA fragment thus obtained as a probe to select positiveclones. The desired gene of I subunit protein of a magnesium chelatasecan be confirmed by determination of the nucleotide sequence of theselected clone.

As the gene encoding D subunit protein of a magnesium chelatase, forexample, those derived from photosynthetic bacterium, Rhodobactersphaeroides (Genebank accession AJ001690), Rhodobacter capsulatus(Geneband accession Z11165), pea (Genebank accession AFO14399), tobacco(Genebank accession Y10022), Synechocystis P.C.C.6803 (Genebankaccession X96599) and the like have been known. The isolation of such aknown gene (its nucleotide sequence has been known) or genes other thanthe above can be carried out in the same manner as described in that ofthe gene encoding I subunit protein of magnesium chelatase.

The genes used in the third aspect of the method of the presentinvention are those encoding proteins having the followingcharacteristics (a) to (c):

(a) having a specific affinity for protoporphyrinogen IX:

(b) having the capability of modifying coproporphyrinogen III; and

(c) being substantially free from framework regions of variable regionsof immunoglobulins.

The term “a specific affinity” for protoporphyrinogen IX in thecharacteristic (a) is substantially the same as that in the above firstor second aspect of the method of the present invention and means thatthe protein and protoporphyrinogen IX bind to each other, enzymaticallyor the protein and protoporphyrinogen IX are bound to each other on thebasis of affinity and specificity as those shown in receptor-chemicalbond such as a bond between a receptor and a ligand and the like. Theproteins may be naturally occurring proteins; variants thereof in whichamino acid substitution, addition, deletion, modification and the likeare introduced into naturally occurring proteins; and artificiallysynthesized proteins having random amino acid sequences which areselected with the guidance of an affinity for protoporphyrinogen IX inso far as they have structures specifically binding toprotoporphyrinogen IX.

The term “having the capability of modifying” coproporphyrinogen III inthe characteristic (b) means that enzymatical reactivity withcoproporphyrinogen III of the proteins is active. For example, thismeans that the protein has the capability of convertingcoproporphyrinogen III into a substance having a chemical structuredifferent from that of coproporphyrinogen III.

The term “substantially free from framework regions of variable regionsof immunoglobulins” means the same as that in the above first or secondaspect of the method of the present invention and the protein does notform the stereostructure specific for the variable regions in theimmunoglobulin as is described hereinabove.

As specific examples of the proteins having the above characteristics(a) to (c), there are active or inactive-type binding proteins having anaffinity for proporphyrinogen IX, for example, active-typecoproporphyrinogen III oxidase whose substrate is proporphyrinogen IX,and the like.

As a reference, the activity of a magnesium chelatase, a ferrochelataseor a coproporphyrinogen III oxidase is, for example, measured by usingthe following method.

(1) A magnesium chelatase:

The genes encoding independent three subunit proteins are used to detecta magnesium chelatase activity according to the method by Gibson, L. C.D., et al. (Proc. Natl. Acad. Sci. USA, 92; p 1941 (1995)) and the like.

(2) A ferrochelatse:

A ferrochelatase activity can, for example, be detected according to themethod by Porra, R. J. (Anal. Biochem., 68; p 289 (1975)) and the like.

(3) A coproporphyrinogen III oxidase:

A coproporphyrinogen III oxidase activity can, for example, be detectedaccording to the method by Yoshinaga, T., Sano, S., et al. (J. Biol.Chem., 255; p 4722 (1980)) and the like.

In the fourth aspect of the method of the present invention, there maybe used in addition to the gene encoding the protein having thecharacteristics (a) to (c) (as described in the first to third aspectsof the present invention), at least one altered form of an enzymaticactivity selected from an altered PPO activity, an altered EPSPSactivity and an altered glyphosate oxidoreductase (GOX) activity. Saidaltered form of the enzymatic activity in the plant cell can give aresistance to a weed control compound in an amount inhibiting anaturally occurring form of said enzymatic activity. Typically, such anamount of the weed control compound is an amount which can set forth aherbicidal control over the growth of a plant cell, which by inhibitinga naturally occurring form of the enzymatic activity. In this regard, togive the resistance to a PPO inhibitory-type herbicidal compound, it ispreferable that the plant cell additionally comprises the altered PPOactivity. Likewise, to give the resistance to glyphosate, it ispreferable that the plant cell additionally comprises the altered EPSPSactivity or the altered GOX activity.

Glyphosate is the common name given to the weed control compound,N-(phosponomethyl)glycine. In this regard, glyphosate includes theammonium salt, sodium salt, isopropylamine salt, trimethylsulfoniumsalt, potassium salt or the like salt. Further, glyphosate is a compoundencompassed by said compound inhibiting EPSPS, as described above.

The term “altered form of an enzymatic activity” means that theenzymatic activity is different from that which naturally occurs in aplant cell, which altered form of an enzymatic activity provides aresistance to a weed control compound that inhibits the naturallyoccurring activity thereof. Said naturally occurring enzymatic activityin the plant cell is the enzymatic activity which occurs naturally inthe absence of direct or indirect manipulation by man of such naturallyoccurring enzymatic activity.

A second gene in the plant cell is useful to confer said altered form ofthe enzymatic activity therein. As such, the second gene typicallyprovides in the plant cell, a gene providing for the altered PPOactivity, for the altered EPSPS activity or for the altered GOXactivity. Various proteins can be encoded by the second gene, so thatthe second gene can provide for said altered form of enzymatic activitywhen expressed in the plant cell.

In utilizing the second gene for the altered PPO activity, the secondgene can encode a naturally occurring protein substantially having PPOactivity. In the plant cell, such a protein substantially having PPOactivity can be a protein having a capability of oxidizingprotoporphyrinogen IX and which has a specific affinity forprotoporphyrinogen IX.

In its amino acid sequence, the protein substantially having PPOactivity preferably contains said region presumed to be FAD binding siteof PPO. Such a protein substantially having PPO activity may be PPO. Asgenes encoding PPO, there can be utilized the known “PPO genes” asdescribed above. Further, there can be utilized a naturally occurringprotein substantially having PPO activity which activity is resistant tothe PPO inhibitory-type herbicidal compound (as described in EP 0770682or WO 9833927).

Further, the protein substantially having PPO activity can havesubstituted, deleted or added thereto amino acids, such that theresulting variant protein has substantially the PPO activity.Conventional methods well known in the art can be used to substitute,delete or add the amino acids thereto. U.S. Pat. No. 5,939,602 and WO9704089 describe variant PPO substantially having PPO activity whichactivity is uninhibited by a PPO inhibitory-type herbicidal compound.The second gene may encode such a variant PPO.

In utilizing the second gene for the altered EPSPS activity, the secondgene can encode a naturally occurring protein substantially having EPSPSactivity. Such a protein substantially having EPSPS activity is aprotein having a capability to modify in the plant cell,phosphoenolpyruvic acid (PEP) with 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimic acid.

Such a protein substantially having EPSPS activity may be EPSPS. As apolynucleotide encoding EPSPS, there can be utilized a knownpolynucleotide encoding EPSPS. Examples of such a polynucleotideencoding EPSPS include those derived from Petunia hybrida (Genebankaccession M37029), Mitchell diploid petunia (as described in EP218571),Salmonella typhymurium (as described in EP508909), Tomato (strain VF36)pistil (Genebank accession M21071), Arabidopsis thaliana (Genebankaccession X06613), soy beans, Zea mays (Genebank accession X63374),Escherichia coli (Genebank accession X00557), Agrobacterium tumefacienssp.strain CP4 (class II) and the like. Additionally, the second gene canalso encode a naturally occurring protein substantially having EPSPSactivity which activity is resistant to glyphosate, such as a bacterialEPSPS which activity is resistant to glyphosate.

Further, the second gene when providing for the altered EPSPS activityin the plant cell can also encode a variant protein substantially havingEPSPS activity. As such, the protein substantially having EPSPS activitycan have substituted, deleted or added thereto amino acids such that theresulting protein substantially has the EPSPS activity. Examples of sucha variant protein substantially having EPSPS activity include a variantEPSPS which activity is resistant to glyphosate, a variant EPSPS inwhich a chloroplast transit peptide is added thereto and the like.

The variant EPSPS which activity is resistant to glyphosate can beproduced by substituting, deleting or adding nucleotides to a geneencoding EPSPS. For example, a substitutive mutation can be introducedto a polynucleotide encoding EPSPS to produce a variant polynucleotide.The protein encoded by the resulting variant gene can then be confirmedfor a resistance to glyphosate and for the EPSPS activity.

The resistance to glyphosate may be confirmed by introducing the variantgene to a particular Escherichia coli mutant and by culturing theresulting particular Escherichia coli mutant in a specified minimalnutrient MOPS medium which has glyphosate added thereto. As theparticular variant Escherichia coli in this case, there is utilized anEscherichia coli mutant which is deficient in its endogenous EPSPS gene(aroA locus) and which has the growth thereof inhibited in the specifiedminimal nutrient MOPS medium (in which there is no glyphosate therein).Further, in this case, the specified minimal nutrient MOPS medium isspecified in that there are no aromatic amino acids present therein.When glyphosate is added to the minimal nutrient MOPS medium, theglyphosate is in an amount which would typically inhibit in normalgrowing conditions, the growth of an Escherichia coli mutant which isdeficient in its endogenous EPSPS gene but which has introduced theretoa naturally occurring gene encoding herbicidally sensitive EPSPS. Byselecting the resulting clones growable in such specified minimalnutrient MOPS medium containing glyphosate, there can be obtained avariant polynucleotide encoding a variant EPSPS having an activity whichis resistant to glyphosate. The EPSPS activity can be confirmed byintroducing said variant gene to a host cell and then by according tothe method described in EP 409815. In this regard, there can be obtainedthe second gene encoding the variant EPSPS substantially having EPSPSactivity which activity is resistant to glyphosate.

In utilizing the second gene for the altered GOX activity, the secondgene can encode a naturally occurring protein substantially having GOXactivity. Such a protein substantially having the GOX activity is aprotein having a capability to degrade glyphosate to less herbicidalproducts, such as aminomethyl phosphonate (AMPA) and glyoxylate. Incases in which glyphosate is degraded into AMPA and glyoxylate, forexample, the protein substantially having GOX activity may cleave theC—N bond of glyphosate.

Such a protein substantially having GOX activity may be GOX. As apolynucleotide encoding GOX, there can be utilized a knownpolynucleotide encoding GOX. Examples of naturally occurring GOX genesinclude those derived from Pseudomonas sp. strains LBAA, Pseudomonas sp.strains LBr, Agrobacterium sp. strain T10 and the like.

Further, the second gene when providing for the altered GOX activity inthe plant cell can encode a variant protein substantially having GOXactivity. As such, the protein substantially having GOX activity canhave substituted, deleted or added thereto amino acids such that theresulting protein substantially has the GOX activity. As an example ofsuch a variant protein substantially having GOX activity, there ismentioned a variant GOX in which a chloroplast transit peptide is addedthereto. Conventional methods well known in the art can be used tosubstitute, delete or add the amino acids thereto.

The GOX activity of a protein substantially having GOX activity can beconfirmed by introducing a gene encoding the protein substantiallyhaving GOX activity into a specified Escherichia coli mutant and byculturing the resulting specified Escherichia coli mutant in a minimalnutrient MOPS medium containing glyphosate as the sole nitrogenoussource therein. As the specified Escherichia coli mutant in this case,there is utilized an Escherichia coli mutant which can grow in a minimalnutrient MOPS medium having a non-herbicidal aminophosphate compound asthe sole nitrogenous source therein, such as E. coli SR2000 Mpu+. Theglyphosate therein is in an amount which would typically inhibit thegrowth of the specified Escherichia coli mutant having no said geneencoding the protein substantially having GOX activity introducedthereto. By selecting the resulting clones growable in such minimalnutrient MOPS medium containing glyphosate as the sole nitrogenoussource therein, there can be obtained a polynucleotide encoding aprotein substantially having GOX activity. Such results suggest that incases in which the protein substantially having GOX activity degradesglyphosate into AMPA, the growable clones use to grow, AMPA as anitrogenous source. In practice, a 3-14C labeled glyphosate may be usedto confirm that said growable clone consumes and degrades glyphosate.For example, the growable clone may be cultured with the 3-14C labeledglyphosate and the cell extract thereof may then be analyzed with HPLC.

The second gene encoding an above protein substantially having PPOactivity, EPSPS activity or GOX activity can be obtained, for example,as follows.

For isolating a known gene encoding a protein substantially having PPOactivity, EPSPS activity or GOX activity, PCR can be carried out byusing genomic DNA or cDNA of an organism having the desired gene as atemplate and primers produced on the basis of nucleotide sequencescorresponding to those about the N- and C-termini of the protein toamplify the desired gene. Further, genes encoding a proteinsubstantially having PPO activity, EPSPS activity or GOX activity can beobtained from organisms other than the above. For example, first, a cDNAlibrary is constructed by obtaining mRNA from an organism andsynthesizing cDNA by using the mRNA as template with reversetranscriptase and integrating the cDNA into a phage vector such as ZAPII, etc. or a plasmid vector such as pUC, etc. For the proteinsubstantially having PPO activity, the cDNA library may be introducedinto Escherichia coli PPO deficient mutant strain VSR800 described byNarita, S., et al., Gene, 182; p 169 (1996), followed by subjecting acomplementation test to select clones containing PPO gene derived fromthe desired organism. Further, for amplifying a DNA fragment containingat least a part of the desired gene, PCR can be carried out by using theabove-constructed cDNA library as a template and primers designed andsynthesized on the basis of nucleotide sequences well conserved amongknown genes such as the above-described genes. Screening of the cDNAlibrary can be carried out by using the DNA fragment thus obtained as aprobe to select positive clones. The desired gene, i.e., a gene encodingthe protein substantially having the PPO activity, EPSPS activity or GOXactivity, can be confirmed by determination of the nucleotide sequenceof the selected clone.

Examples of methods used to confer the altered EPSPS activity or alteredGOX activity include the following. An example may include a method ofintroducing into a cultivated plant a gene having a polynucleotidesequence encoding a petunia (Mitchell diploid petunia) EPSPS downstreamof a high expression promoter such as a cauliflower mosaic virus 35Spromoter (EP 218571). A further example may include a method ofintroducing into a cultivated plant a gene having a 35S promoterupstream of a polynucleotide sequence encoding an Agrobacterium(Agrobacterium tumefaciens sp. strain CP4) EPSPS fused with achloroplast transit peptide of a petunia (Petunia hybrida) EPSPS (WO0204449, U.S. Pat. No. 5,633,435). A furthermore example may include amethod of introducing into a cultivated plant a gene having 2 continuous35S promoters upstream a polynucleotide encoding a sunflower chloroplasttransit peptide of small subunit of ribulose-1,5-bisphosphatecarboxylase (ssRUBISCO), the 22 amino acids from the N-terminus of maizessRUBISCO, maize chloroplast transit peptide of ssRUBISCO and aSalmonella (Salmonella typhyrium) EPSPS (EP 508909). Even furthermore,an example may include a method of introducing into a cultivated plant,a gene having downstream from a promoter of Arabidopsis thaliana alcoholdehydrogenase A, a polynucleotide encoding an Arabidopsis thalianachloroplast transit peptide and GOX (WO 9706269). Yet even furthermore,an example may include a method of introducing into a cultivated plant,the above gene encoding GOX as well as a gene having downstream from a35S promoter possessing an enhanced promoter activity with the omegasequence of tobacco mosaic virus, a polynucleotide sequence whichencodes an Agrobacterium (Agrobacterium tumefaciens sp. strain CP4)EPSPS (class II) downstream a chloroplast transit peptide of Petunia(Petunia hybrida) EPSPS (WO 9706269). Still yet even furthermore, anexample may include a method of introducing into a cultivated plant, agene encoding a variant EPSPS having amino acid substitutions thereinwhich augment the resistant to glyphosate [Minchee, M. A. W. et al.,BIO/TECHNOLOGY, 6: p 915 (1988), EP 389066, EP 409815, WO 9206201 andU.S. Pat. No. 5,312,910].

Examples of methods used to confer the altered PPO activity include thefollowing. An example may include a method of over-expressing in a plantcell, a gene encoding wild-type, naturally occurring PPO) (U.S. Pat. No.5,767,373). A further example may include a method of expressing in aplant cell, a variant protein substantially having PPO activity whichactivity is not inhibited by a PPO inhibitory-type herbicidal compound(U.S. Pat. No. 5,939,602). A furthermore example may include a method ofexpressing a PPO substantially having PPO activity which is notinhibited by a PPO inhibitory-type herbicidal compound, wherein the PPOis derived from bacteria (EP 0770682 or WO 9833927).

In the method (including the above first to third aspects) of thepresent invention, for introducing the gene encoding the protein havingthe characteristics of (a) to (c) into a plant cell, a gene encoding oneprotein can be introduced. Further, plural genes encoding differentproteins can be introduced into a plant cell. When said altered form ofenzymatic activity is given to the plant cell, the second gene encodingone protein may also be introduced. Further, plural genes of the secondgene can be introduced into the plant cell to provide for said alteredform of enzymatic activity therein. In introducing the gene encoding theprotein having the characteristics of (a) to (c) and second genethereto, the gene encoding the protein having the characteristics of (a)to (c) may be introduced into the plant cell with the second gene, ormay be introduced before or after the second gene is introduced to theplant cell. Such gene introduction into plant cells can be carried outby conventional gene engineering techniques, for example, Agrobacteriuminfection (JP-B 2-58917 and JP-A 60-70070), electroporation intoprotoplasts (JP-A 60-251887 and JP-A 5-68575), particle gun methods(JP-A 5-508316 and JP-A 63-258525), and the like.

Preferably, the gene to be introduced into a plant cell is integratedinto a vector having a selection marker gene such as a gene which cangive cell growth inhibitor resistance to the plant cell, for example,the gene encoding the protein having live characteristics of (a) to (c)and the second gene, when utilized for the altered form of enzymaticactivity, can be integrated into one of such vectors. Further, the geneencoding the protein having the characteristics of (a) to (c) and thesecond gene may also each be integrated, respectively, into such vectorshaving a selection marker gene. In integrating the gene encoding theprotein having the characteristics (a) to (c) and the second gene intosuch respective vectors, the selection marker gene utilized for thevector for the second gene is typically different from the selectionmarker gene utilized for the vector for the gene encoding the proteinhaving the characteristics (a) to (c).

For expression of the gene encoding the protein having thecharacteristics (a) to (c) in the plant cell, the gene can be introducedinto a chromosome of a plant cell by homologous recombination [Fraley,R. T. et al., Proc. Natl. Acad. Sci. USA, 80; p 4803 (1983)] to selectthe plant cell expressing the gene. Alternatively, the gene can beintroduced into a plant cell in the form that it is operably ligated toa promoter and a terminator both of which can function in the plantcell.

The term “operably ligated” used herein means that the above promoterand terminator are joined in such a state that the introduced gene isexpressed in the plant cell under control of the promoter and theterminator.

To provide for the altered form of enzymatic activity, the second geneis expressed in a plant cell. For expression of the second gene in theplant cell, the second gene can likewise be introduced into a chromosomeof a plant cell by homologous recombination to select the plant cellexpressing the second gene. Alternatively, the second gene can beintroduced into a plant cell in the form that it is operably ligated toa promoter and a terminator both of which can function in the plantcell. When utilized, the second gene is typically expressed at a levelsuch that the amount of the protein encoded by the second gene providesfor the altered form of enzymatic activity and further confer theresistance of the plant cell. It is preferable when the second geneencodes PPO or EPSPS, that the second gene provide for the altered formof enzymatic activity through over-expression. If so desired, atranscriptionally strong promoter which can function in the plant cellcan be utilized with the second gene.

As the promoter which can function in a plant cell, for example, thereare constitutive promoters derived from T-DNA such as nopaline synthasegene promoter, octopine synthase gene promoter, etc., promoters derivedfrom plant viruses such as 19S and 35S promoters derived fromcauliflower mosaic virus, etc., inductive promoters such asphenylalanine ammonia-lyase gene promoter, chalcone synthase genepromoter, pathogenesis-related protein gene promoter, etc., and thelike. The promoter is not limited these promoters and other plantpromoters can be used.

As the terminator which can function in a plant cell, for example, thereare terminators derived from T-DNA such as nopaline synthase terminator,etc., terminators derived from plant viruses such as terminators derivedfrom garlic viruses GV1, GV2, etc., and the like. The terminator is notlimited to these terminators and other plant terminators can be used.

As the plant cells into which the gene encoding the protein having thecharacteristics of (a) to (c) are introduced, for example, there areplant tissues, whole plants, cultured cells, seeds and the like.Examples of the plant species into which the genes are introducedinclude dicotyledones such as tobacco, cotton, rapeseed, sugar beet,mouse-ear cress, canola, Max, sunflower, potato, alfalfa, lettuce,banana, soybean, pea, legume, pine, poplar, apple, grape, citrus fruits,nuts, etc.; and monocotyledones such as corn, rice, wheat, barley, rye,oat, sorghum, sugar cane, lawn, etc. The second gene may also beintroduced into such plant cells.

The transformant plant cells expressing the gene encoding the proteinhaving the characteristics of (a) to (c) can be obtained by culturingcells into which the gene is transferred in a selection culture mediumcorresponding to a selection marker joined to the locus on the gene, forexample, a culture medium containing a cell growth inhibitor, or thelike, and isolating a clone capable of growing in the culture medium.Further, the selection culture medium should also correspond to aselection marker joined to the locus of the second gene when the alteredform of enzymatic activity is also present in the transformant plantcells. Alternatively, the above transformant plant cells can be selectedby culturing plant cells into which the gene is introduced in a culturemedium containing the weed control compound to which the resistance isgiven, and isolating clones capable of growing in the culture medium.

The desired weed control compound-resistant plant can be obtained fromthe transformant cells thus obtained by regenerating the whole plantaccording to a conventional plant cell culture method, for example, thatdescribed in Plant Gene Manipulation Manual, Method for ProducingTransgenic Plants, UCHIMIYA, Kodansha Scientific (1996). Thus, thetransformed plants such as plant tissues, whole plants, cultured cells,seeds and the like can be obtained.

For example, rice and mouse-ear cress expressing the gene encoding theprotein having the characteristics of (a) to (c) can be obtainedaccording to the method described Experimental Protocol of Model Plants,Rice and Mouse-Ear Cress Edition, (Supervisors: Koh SHIMAMOTO andKiyotaka OKADA, Shujun-sha, 1996), Chapter 4. Further, according to themethod described in JP-A 3-291501, soybean expressing the gene encodingthe binding protein by introducing the gene into soybean adventitiousembryo with a particle gun. Likewise, according to the method describedby Fromm, M. E., et al., Bio/Technology, 8; p 838 (1990), cornexpressing the gene encoding the above protein can be obtained byintroducing the gene into adventitious embryo with a particle gun. Wheatexpressing the gene encoding the above protein can be obtained byintroducing the gene into sterile-cultured wheat immature scutellum witha particle gun according to a conventional method described by TAKUMI etal., Journal of Breeding Society (1995), 44: Extra Vol. 1, p 57.Likewise, according to a conventional method described by HAGIO, et al.,Journal of Breeding Society (1995). 44; Extra Vol. 1, p 67, barleyexpressing the gene encoding the above protein can be obtained byintroducing the gene into sterile-cultured barley immature scutellumwith a particle gun.

For confirmation of weed control compound-resistance of the plantexpressing the gene encoding the above protein, preferably, the plant isreproduced with applying the weed control compound to which resistanceis given to evaluate the degree of reproduction of the plant. For morequantitative confirmation, for example, in case of resistance to acompound having PPO inhibitory-type herbicidal activity, preferably,pieces of leaves of the plant are dipped in aqueous solutions containingthe compound having PPO inhibitory-type herbicidal activity at variousconcentrations, or the aqueous solutions containing the compound havingherbicidal activity are sprayed on pieces of leaves of the plant,followed by allowing to stand on an agar medium in the light at roomtemperature. After several days, chlorophyll is extracted from the plantleaves according to the method described by Mackenney, G., J. Biol.Chem., 140; p 315 (1941) to determine the content of chlorophyll.

Since the weed control compound-resistant plants (e.g., plant tissues,whole plants, cultured cells, seeds, etc.) obtained by the method of thepresent invention (including the first to fourth aspects) showresistance to weed control compounds, even in case that a weed controlcompound is applied to a growth area (e.g., cultivation area,proliferation area, etc.), the plant can grow. Therefore, when a weedcontrol compound is applied to a growth area of the desired weed controlcompound resistant-plant, the desired plant can be protected from plantswithout resistance to the weed control plant. For example, weeds can becontrolled efficiently by applying a weed control compound on a growtharea of the plant having resistance to the weed control compound.

Further, by applying a weed control compound to a growth area of theweed control, compound-resistant plant obtained by the method of thepresent invention (including the first to third aspects) and otherplants (e.g., those having no or weak resistance to the weed controlcompound), one of the plants can be selected on the basis of thedifference in growth between the plants. For example, by applying(adding) a weed control compound to a cultivation area (culture medium)of the weed control compound-resistant plant cells obtained by themethod of the present invention and other plant cells (e.g., thosehaving no or weak resistance to the weed control compound), one of theplant cells can be selected efficiently on the basis of the differencein growth between the plants.

The following Examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

EXAMPLE 1

Isolation of Protoporphyrin IX Binding Subunit Protein Gene of MagnesiumChelatase.

Genomic DNA of photosynthetic bacterium Rhodobacter sphaeroidesATCC17023 was prepared using ISOPLANT kit for genomic DNA preparation(manufactured by Nippon Gene). Then, according to the description ofGibson, L. C. D. et al., Proc. Natl. Acad. Sci. USA, 92; p 1941 (1995),PCR was carried out by using about 1 μg of said genomic DNA as atemplate, and 10 pmol of an oligonucleotide composed of nucleotidesequence represented by SEQ ID NO: 1 and 10 pmol of an oligonucleotidecomposed of nucleotide sequence represented by SEQ ID NO: 2 as primersto amplify the DNA fragment containing protoporphyrin IX binding subunitprotein gene bchH of magnesium chelatase. The oligonucleotides wereprepared with a DNA synthesizer (PE Applied Biosystems: Model 394DNA/RNA Synthesizer) and purified with an oligonucleotide purificationcartridge (PE Applied Biosystems: OPC Cartridge). The PCR was carriedout by maintaining at 94° C. for 2 minutes, at 96° C. for 40 seconds andthen at 68° C. for 7 minutes, repeating a cycle for maintaining at 96°C. for 40 seconds and then at 68° C. for 7 minutes 28 times, and finallymaintaining at 96° C. for 40 seconds, at 68° C. for 7 minutes and thenat 72° C. for 10 minutes.

EXAMPLE 2

Expression of Protoporphyrin IX Binding Subunit Protein Gene ofMagnesium Chelatase in Escherichia Coli (Hereinafter Abbreviated to E.coli).

According to the description of Gibson, L. C. D. et al., Proc. Natl.Acad. Sci. USA, 92; p 1941 (1995), the DNA fragment containing bchH geneprepared in Example 1 was digested with the restriction enzymes NdeI andBglII. The resultant DNA fragment was inserted between NdeI restrictionsite and BamHI restriction site of expression vector pET11a(manufactured by Stratagene) to obtain plasmid pETBCH (FIG. 1). Thisplasmid pETBCH was introduced into E. coli BL21(DE3) strain competentcells (manufactured by Stratagene) according to the manual attached tothe competent cells to obtain E. coli BL21(DE3)/pETBCH strain. Thestrain was inoculated into 1.5 ml LB liquid culture medium (1% tryptone,0.5% yeast extract, 0.5% NaCl) containing 100 μg/ml ampicillin in a tube(14×10 mm), and the tube was covered with aluminum foil (hereinafterreferred to as dark conditions), cultured with shaking at 37° C. underlight of fluorescent lamp (about 8000 lux). When the absorbance at 600nm of the liquid culture medium became about 0.6, isopropylβ-D-thiogalactopyranoside (IPTG) was added to the liquid culture mediumso that the final concentration was 0.4 mM, and the culture wascontinued for about additional 20 hours. At that time, the Escherichiacoli turned red and fluorescent absorbance (excitation wavelength 405nm, emission wavelength 630 nm) which showed the accumulation ofprotoporphyrin IX in E. coli was observed. When E. coli BL21(DE3)/pETBCHstrain was cultured according to the same manner except that IPTG wasnot added, E. coli did not turned red and the above fluorescentabsorbance did not detected. In contrast to this, when E. coliBL21(DE3)/pETBCH strain was cultured according to the same manner (withIPTG) except that the tube was not covered with aluminum foil(hereinafter referred to as light conditions), E. coli grew and turnedred as above.

EXAMPLE 3

Expression of PPO Gene Derived from Soybeans in hemG Gene Deficient E.coli.

Soybeans (Glycine max var. Williams82) were seeded and cultivated at 25°C. for 20 days and green leaves were collected. The collected greenleaves were frozen with liquid nitrogen and the frozen leaves wereground in a mortar with a pestle. From the ground leaves, RNA wereextracted by using RNA extracting reagent ISOGEN (manufactured by NipponGene) according to the manual attached thereto. The resultant RNA liquidextract was subjected to ethanol precipitation to collect total RNA,then the total RNA was fractionated by using poly (A) RNA fractionatingkit BIOMAG mRNA Purification Kit (manufactured by Perceptive Bio System)according to the manual attached thereto to collect poly (A) RNAfraction. Using 1 μg of this poly (A) RNA fraction as a template, cDNAwas synthesized with the cDNA synthetic reagent contained in MarathoncDNA amplification kit (manufactured by Clontech) according to themanual attached thereto. PCR was carried out by using the resultant cDNAas a template, and an oligonucleotide composed of nucleotide sequence ofSEQ ID NO: 3 and an oligonucleotide composed of nucleotide sequence ofSEQ ID NO: 4 as primers to amplify the DNA fragment containingchloroplast-type protoporphyrinogen IX oxidase gene. The aboveoligonucleotides were prepared with a DNA synthesizer (PE AppliedBiosystems: Model 394 DNA/RNA Synthesizer) and purified with anoligonucleotide purification cartridge (PE Applied Biosystems: OPCCartridge). The PCR was carried out by maintaining at 94° C. for 1minutes and then at 65° C. for 5 minutes, repeating a cycle formaintaining at 94° C. for 15 seconds and then at 65° C. for 5 minutes 29times. After the PCR, the amplified DNA fragment was purified byfiltering the reaction mixture with MicroSpin S-400HR (manufactured byPharmacia Biotech), and the DNA fragment was ligated to plasmid pCR2.1(manufactured by Invitrogen) cleaved by restriction enzyme Sail toobtain plasmid pSPPO-P. Then, the plasmid was introduced into competentcells of E. coli INVαF′ strain (manufactured by Invitrogen) andampicillin resistant strains were selected. Then, the plasmid containedin selected ampicillin resistant strains was sequenced by using Dyeterminator cycle sequencing kit (manufactured by PE Applied Biosystems)and DNA sequencer 373S (manufactured by PE Applied Biosystems). As aresult, the nucleotide sequence of SEQ ID NO: 5 was revealed, therebyconfirming that plasmid pSPPO-P contained chloroplast-typeprotoporphyrinogen IX oxidase gene of soybean.

The plasmid pSPPO-P was digested with restriction enzyme PshBI, theresultant DNA fragment was blunted by using T4 DNA polymerase andfurther digested with SphI to isolate the DNA fragment containingchloroplast-type PPO gene of soybean and lac promoter. Then, the plasmidpACYC184 (manufactured by Nippon Gene) was digested with the restrictionenzymes NruI and SphI to remove a fragment of 410 bp and the above DNAfragment was inserted instead to obtain plasmid pACYCSP (FIG. 2). Then,the plasmid pACYCSP was introduced into PPO gene (hemG gene locus)deficient mutant E. coli BT3 strain (described in Yamamoto, F. et al.,Japanese J. Genet., 63; p 237 (1988) etc.) according to the methoddescribed in Manahan, D. J., Mol. Biol., 166, p 557 (1983). Theresultant E. coli were cultured in YPT medium (5 g/liter yeast extract,5 g/liter tryptone, 5 g/liter peptone, 10 g/liter NaCl, pH 7.0)containing 15 μg/ml chloramphenicol and 10 μg/ml kanamycin to select E.coli BT3/pACYCSP strain resistant to chloramphenicol and kanamycin whosehemG gene deficiency was complemented by PPO gene derived from soybean.

EXAMPLE 4

Test of Protoporphyrin IX Binding Subunit Protein of Magnesium Chelatasefor Capability of Giving Weed Control Compound-Resistance

E. coli BT3/pACYCSP strain prepared in Example 3 was inoculated into YPTmedium containing 10 or 1 ppm of PPO inhibitory type herbicidal compoundrepresented by the above Structure 8, 10 μg/ml hemin, 50 μg/mlaminolevulinic acid, 15 μg/ml chloramphenicol and 10 μg/ml kanamycin,cultured under dark conditions or light conditions according to the samemanner as in Example 2. As a control, E. coli BT3/pACYCSP strain wascultured in the same medium as above without the herbicidal compoundsunder the same conditions. Then, 18 hours after initiation of culture,the absorbance of the liquid culture medium was measured at 600 nm. Bytaking the absorbance of the medium without the herbicidal compound as1, the relative value of the absorbance of the medium containing theherbicidal compound was calculated. The results are shown in Table 1.

TABLE 1 Relative absorbance Concentration of E. coli Culture testcompound strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP in the light0.10 0.25 1.0 BT3/pACYCSP in the dark 0.73 0.95 1.0

Plasmid pTVBCH (FIG. 3) was constructed by amplification of the DNAfragment containing bchH gene derived from photosynthetic bacteriumRhodobacter sphaeroides using the oligonucleotide composed of thenucleotide sequence of SEQ ID NO: 1 and the oligonucleotide composed ofthe nucleotide sequence of SEQ ID NO: 2 according to the same manner asin Example 1, digestion of the resultant DNA fragment with therestriction enzymes NcoI and BglII and introducing the digested DNAfragment between NcoI restriction site and BamHI restriction site ofplasmid pTV118N (manufactured by Takara Shuzo Co., Ltd.).

Plasmids pTVBCH and pTV118N respectively were introduced into E. coliBT3/pACYCSP strain prepared in Example 3 according to the methoddescribed in Hanahan, D. J., Mol. Biol., 166; p 557 (1983). Theresultant E. coli were cultured in YPT medium containing 100 μg/mlampicillin, 15 μg/ml chloramphenicol and 10 μg/ml kanamycin to obtain E.coli BT3/pACYCSP+pTVBCH strain bearing plasmids pACYCSP and pTVBCH, andE. coli BT3/pACYCSP+pTV118N strain bearing plasmids pACYCSP and pTV118N.

These strains were inoculated into YPT medium containing 10 or 1 ppm ofthe PPO inhibitory-type herbicidal compound represented by the aboveStructure 8, 100 μg/ml ampicillin, 15 μg/ml chloramphenicol, 10 μg/mlkanamycin, 10 μg/ml hemin and 50 μg/ml aminolevulinic acid, culturedunder dark conditions or light conditions according to the same manneras in Example 2. Then, 18 hours after initiation of culture, theabsorbance of the liquid culture medium was measured at 600 nm. Bytaking the absorbance of the medium without the herbicidal compound as1, the relative value of the absorbance of the medium containing theherbicidal compound was calculated. The results are shown in Table 2.

TABLE 2 Relative absorbance Concentration of test Culture compound E.coli strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP + pTVBCH in thelight 0.80 0.77 1.0 BT3/pACYCSP + pTVBCH in the dark 0.90 1.06 1.0BT3/pACYCSP + pTV118N in the light 0.18 0.31 1.0 BT3/pACYCSP + pTV118Nin the dark 0.68 0.77 1.0

Further, these strains were inoculated into YPT medium containing PPOinhibitory-type herbicidal compounds represented by the above Structures1, 14, 15, 18-22, 29, 32, 33, 34 and 36, respectively, 100 μg/mlampicillin, 15 μg/ml chloramphenicol, 10 μg/ml kanamycin, 10 μg/ml heminand 50 μg/ml aminolevulinic acid, cultured under dark conditions orlight conditions similar to the Example 2. Then, 18 hours afterinitiation of culture, the absorbance of liquid culture medium wasmeasured at 600 nm. By taking the absorbance of the medium without theherbicidal compound as 1, the relative value of the absorbance of themedium containing the herbicidal compound was calculated. The resultsare shown in Table 3.

TABLE 3 Relative absorbance Test compound Test BT3/pACYCSP + pTVBCHBT3/pACYCSP + pTV118N Structure No. concentration in the light in thedark in the light in the dark Structure 1 5.0 0.88 0.88 0.31 0.87Structure 14 10 0.47 0.93 0.12 0.81 Structure 15 0.5 0.94 0.94 0.38 0.82Structure 18 2.0 0.68 1.0 0.33 0.91 Structure 19 5.0 0.78 0.89 0.40 0.71Structure 20 5.0 0.57 0.88 0.11 0.75 Structure 21 10 0.88 0.91 0.25 0.85Structure 22 10 0.55 0.93 0.29 0.94 Structure 29 0.5 0.64 0.90 0.22 0.77Structure 32 2.0 0.70 0.94 0.37 0.87 Structure 33 2.0 0.81 0.92 0.410.91 Structure 34 1.0 0.41 0.94 0.19 0.86 Structure 36 0.5 0.55 0.950.28 0.96

EXAMPLE 5

Introduction of Gene Encoding Protoporphyrin IX Binding Subunit Proteinof Magnesium Chelatase into Tobacco

A plasmid was constructed for introducing bchH gene into a plant byAgrobacterium infection method. First, binary vector pBI121(manufacturedby Clontech) was digested with restriction enzyme SacI, and KpnI linker(manufactured by Takara Shuzo Co., Ltd.) was inserted to prepare plasmidpBIK wherein Sad recognition site of pBI121 was removed and KpnIrecognition site was added. On the other hand, according to the samemanner as described in Example 1, PCR was carried out by using thegenomic DNA of photosynthetic bacterium Rhodobacter sphaeroides as atemplate, and the oligonucleotide primer composed of the nucleotidesequence of SEQ ID NO: 7 and the oligonucleotide primer composed of thenucleotide sequence of SEQ ID NO: 8 to amplify the DNA fragmentcontaining bchH gene. Then, the above plasmid pBIK was digested withrestriction enzymes XbaI and KpnI to remove β-glucuronidase gene, andinstead thereof, a DNA fragment which was obtained by digesting theabove DNA fragment containing bchH gene with restriction enzymes XbaIand KpnI was inserted to produce plasmid pBIBCH (FIG. 4) in which bchHgene was joined downstream from 35S promoter. Binary vector pBI121(manufactured by Clontech) was also digested with restriction enzymesBamHI and ScaI to remove β-glucuronidase gene, the resultant DNAfragment was blunted by using T4 DNA polymerase, followed byself-cyclization with T4 DNA ligase to construct plasmid pNO (FIG. 5).The plasmid was used as a vector control of bchH expression plasmidpBIBCH.

The plasmid pBIBCH and pNO were introduced into Agrobacteriumtumefaciens LBA4404, respectively. Agrobacterium strain bearing pBIBCHand that bearing pNO were isolated by culturing the resultanttransformants in a medium containing 300 μg/ml streptomycin, 100 μg/mlrifampicin and 25 μg/ml kanamycin and selecting the desiredtransformants.

Then, according to the method described in Manual for Gene Manipulationof Plant (by Hirofumi UCHIMIYA, Kodan-sha Scientific, 1992), the genewas introduced into tobacco. Agrobacterium strain bearing plasmid pBIBCHwas cultured at 28° C. overnight in LB medium and then leaf pieces oftobacco cultured sterilely were dipped in the liquid culture medium. Theleaf pieces were cultured at room temperature for 2 days inMurashige-Skoog medium (MS-medium, described in Murasige T. and SkoogF., Physiol. Plant. (1962) 15, p 473) containing 0.8% agar, 0.1 mg/liternaphthalene acetic acid and 1.0 mg/liter benzyl aminopurine. Then, theleaf pieces were washed with sterilized water and cultured for 7 days onMS medium containing 0.8% agar, 0.1 mg/liter naphthalene acetic acid,1.0 mg/liter benzyl aminopurine and 500 μg/ml cefotaxime. The leafpieces were transplanted onto MS medium containing 0.8% agar, 0.1mg/liter naphthalene acetic acid, 1.0 mg/liter benzyl aminopurine, 500μg/ml cefotaxime and 100 μg/ml kanamycin (hereinafter referred to asselective MS medium) and cultured on the medium continuously for 4monthswith transplanting the tobacco leaf pieces onto fresh selective MSmedium every 1 month. During culture, stem-leaf differentiated shootswere appeared from the tobacco leaf pieces, these shoots weretransplanted to MS medium containing 0.8% agar, 300 μg/ml cefotaxime and50 μg/ml kanamycin to induce roots to obtain regenerated plants. Theresultant regenerated plant was transplanted and cultured on MS medium0.8% agar and 50 μg/ml kanamycin to obtain tobacco plant into which bchHgene was introduced. Similarly, tobacco leaf pieces were infected withAgrobacterium strain bearing pNO to obtain regenerated plant from thetobacco leaf pieces and tobacco plant (hereinafter referred to ascontrol recombinant tobacco).

EXAMPLE 6

Test of Tobacco Bearing Introduced Gene Encoding Protoporphyrin IXBinding Subunit Protein of Magnesium Chelatase for Resistance toHerbicidal Compounds

The tobacco leaves into which bchH gene was introduced and controlrecombinant tobacco leaves obtained in Example 5 were collected and eachleaf was divided into the right and left equivalent pieces along themain vein, respectively. To one piece was applied an aqueous solutioncontaining 0.3 ppm PPO inhibitory-type herbicidal compound of Structure8, while, to the other piece was not applied the compound. These leafpieces were placed on MS medium containing 0.8% agar and allowed tostand at room temperature for 7 days in light place. Then, each leafpiece was ground with pestle and mortar in 5 ml of 80% aqueous acetonesolution to extract chlorophyll. The extract liquid was diluted with 80%aqueous acetone solution and the absorbance was measured at 750 nm, 663nm and 645 nm to calculate total chlorophyll content according to themethod described by Macknney G., J. Biol. Chem. (1941) 140, p 315. Theresults obtained from 4 clones of tobacco into which bchH gene wasintroduced (BCH1 to 4) and control recombinant tobacco is shown in Table4. In the table, the resistant level to the herbicidal compound wasrepresented by percentages of the total chlorophyll content of leafpieces treated with herbicidal compound to that of untreated leafpieces.

TABLE 4 Total chlorophyll content (mg/g-fresh weight) ResistantRecombinant untreated- level to test tobacco leaf treated-leaf compound(%) control 2.49 0.19 7.63 BCH-1 1.35 1.70 126 BCH-2 2.06 2.14 104 BCH-31.93 1.57 81.3 BCH-4 1.51 1.06 70.2

The tobacco clone into which bchH gene was introduced and controlrecombinant tobacco were also treated in the same manner with thesolution containing PPO inhibitory-type herbicidal compound representedby the above Structure 3, 7, 10, 11, 13, 17, 23, 24, 25, 27, 28, 30 or35, and the resistant level to each herbicidal compound was measured.The results are shown in Table 5. In the table, the resistant levels tothe herbicidal compound were represented by percentages of the totalchlorophyll content of leaf pieces treated with the herbicidal compoundto that of untreated leaf pieces.

TABLE 5 Resistant level to test Test compound (%) compound Test bchHcontrol Structure concentration recombinant recombinant No. (ppm)tobacco tobacco Structure 3 10 114 9.94 Structure 7 30 89.3 8.62Structure 10 10 84.0 14.9 Structure 11 0.30 78.1 5.51 Structure 13 3095.2 14.8 Structure 17 0.30 80.4 14.3 Structure 23 3.0 106 5.58Structure 24 10 129 5.18 Structure 25 10 104 16.0 Structure 27 10 86.816.8 Structure 28 0.30 72.2 8.79 Structure 30 3.0 102 4.24 Structure 350.30 83.3 17.4

EXAMPLE 7

Isolation of Gene Encoding Variant Protein of Protoporphyrin IX BindingSubunit Protein of Tobacco Magnesium Chelatase

Total RNAs were prepared from leaf tissues of tobacco (Nicotiana tabacumcv. SR1) by using RNeasy Plant Kit (manufactured by QIAGEN) according tothe manual attached thereto. The DNA fragment containing the geneencoding protoporphyrin IX binding subunit protein of tobacco magnesiumchelatase whose chloroplast transit signal had been deleted (hereinafterreferred to as the variant tobacco chelatase subunit) was obtained byusing RNA LA PCR Kit (AMV) Ver 1.1 (manufactured by Takara Shuzo Co.,Ltd.) according to the manual attached thereto. First, 1st strand cDNAwas synthesized by using tobacco total RNAs as templates and OligodT-Adaptor Primer contained in the above kit as the primer with thereverse transcriptase contained in the above kit. Then, PCR was carriedout by using the 1st strand cDNA as a template and LA Taq polymerasecontained in the above kit to amplify the DNA fragment containing thegene encoding the variant tobacco chelatase subunit protein. In this PCRoligonucleotide primer composed of the nucleotide sequence of SEQ ID NO:9 and the oligonucleotide primer composed of the nucleotide sequence ofSEQ ID NO: 10 were used. These oligonucleotides were synthesized byusing a DNA synthesizer (PE Applied Biosystems; Model 394 DNA/RNASynthesizer) and purified with an oligonucleotide purification cartridge(PE Applied Biosystems; OPC cartridge). The PCR was carried out bymaintaining at 94° C. for 2 minutes and then repeating a cycle formaintaining at 94° C. for 30 seconds, at 50° C. for 30 seconds and thenat 72° C. for 7 minutes 30 times. After the PCR, the DNA fragmentamplified by the PCR was cloned into plasmid pCR2.1 by using TA CloningKit (manufactured by Invitrogen) according to the manual attachedthereto. The resultant plasmid was digested with restriction enzyme KpnIand analyzed by agarose gel electrophoresis. The plasmid from which 8.0kb DNA fragment was detected was named pTCHLH. The plasmid had thestructure that the gene encoding the variant tobacco chelatase subunithas been inserted in the direction expressible under the control of lacpromoter. Plasmid pTCHLH was digested with restriction enzyme KpnIfollowed by self-ligation to obtain plasmid pTCHLH1 (FIG. 6) in whichDNA fragment composed of about 60 nucleotides had been deleted fromplasmid pTCHLH.

EXAMPLE 8

Test of Variant Tobacco Magnesium Chelatase Subunit Protein forCapability of Giving Resistance to Herbicidal Compounds

The plasmid pTCHLH1 and pCR2.1 prepared in Example 7 were introducedinto E. coli BT3/pACYCSP strain prepared in Example 3, respectivelyaccording to the method described in Hanahan, D. J., Mol. Biol., 166; p557 (1983). E. coli BT3/pACYCSP+pTCHLH1 strain bearing plasmids pACYCSPand pTCHLH1, and E. coli BT3/pACYCSP+pCR2.1 strain bearing plasmidspACYCSP and pCR2.1 were obtained by culturing the above strains in YPTmedium containing 100 μg/ml ampicillin, 15 μg/ml chloramphenicol and 50μg/ml kanamycin, respectively.

These E. coli strains were inoculated into YPT medium containing 10 or 1ppm of the PPO inhibitory-type herbicidal compound represented byStructure 8, 100 μg/ml ampicillin, 15 μg/ml chloramphenicol, 50 μg/mlkanamycin, 10 μg/ml hemin and 50 μg/ml aminolevulinic acid, culturedunder dark conditions or light conditions according to the same manneras in Example 2. Then, 18 hours after initiation of culture, theabsorbance of the liquid culture medium was measured at 600 nm. Bytaking the absorbance of the medium without the herbicidal compound as1, the relative value of the absorbance of the medium containing theherbicidal compound was calculated. The results are shown in Table 6.

TABLE 6 Relative absorbance concentration of test Culture compound E.coli strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP + pTCHLH1 in thelight 0.69 0.89 1.0 BT3/pACYCSP + pTCHLH1 in the dark 0.92 0.93 1.0BT3/pACYCSP + pCR2.1 in the light 0.03 0.08 1.0 BT3/pACYCSP + pCR2.1 inthe dark 1.0 1.0 1.0

EXAMPLE 9

Introduction of Gene Encoding Variant Tobacco Magnesium ChelataseSubunit Protein into Tobacco

A plasmid for introducing the gene encoding a variant tobacco magnesiumchelatase subunit protein into tobacco by Agrobacterium infection methodwas constructed. First, the DNA fragment containing the gene encodingthe variant tobacco magnesium chelatase subunit protein was prepared bydigesting plasmid pTCHLH1 prepared in Example 7 with restriction enzymesKpnI and SalI. On the other hand, binary vector pBI121 (manufactured byClonetech) was digested with restriction enzyme SmaI and KpnI linker(manufactured by Takara Shuzo Co., Ltd.) was inserted into this portionto prepare plasmid pBI121K in which SmaI recognition site of pBI121 wasremoved and KpnI recognition site was added. The plasmid pBI121K wasdigested with restriction enzyme SacI followed by blunting the DNA byadding nucleotides to the double-stranded DNA gap with DNA Polymerase I.Then, the DNA was dephosphorylated with alkaline phosphatase derivedfrom calf intestine and cyclized by inserting phosphorylated Sail linker(4680P, manufactured by Takara Shuzo Co., Ltd.) to construct plasmidpBI121KS. The binary vector pBI121KS was digested with restrictionenzymes KpnI and SalI to remove β-glucuronidase gene and the geneencoding the variant tobacco magnesium chelatase subunit protein wasinserted into this portion to prepare plasmid pBITCHLH (FIG. 7).

The plasmid pBITCHLH was introduced into Agrobacterium tumefaciensLBA4404. The resultant transformants were cultured in a mediumcontaining 300 μg/ml streptomycin, 100 μg/ml rifampicin and 25 μg/mlkanamycin, followed by selecting the desired transformants to isolate aAgrobacterium strain bearing pBITCHLH.

Leaf pieces of tobacco cultured sterilely are infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco into which the gene encoding the variant tobacco magnesiumchelatase subunit protein is introduced is obtained.

EXAMPLE 10

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Gene Encoding Variant Tobacco Magnesium Chelatase SubunitProtein

The levels of resistance to herbicidal compounds are confirmedquantitatively by testing tobacco introduced with the gene encoding thevariant tobacco magnesium chelatase subunit protein prepared in Example9 according to the same manner as in Example 6.

EXAMPLE 11

Isolation of Gene Encoding Variant Protein of Soybean PPO Having NoCapability of Oxidizing Protoporphyrinogen IX and Having SpecificAffinity for Protoporphyrinogen IX

PCR was carried out by using plasmid pSPPO-P prepared in Example 3 as atemplate and an oligonucleotide composed of the nucleotide sequence ofSEQ ID NO: 11 and an oligonucleotide composed of the nucleotide sequenceof SEQ ID NO: 12 as primers to amplify the DNA fragment encoding soybeanPPO whose chloroplast transit signal and FAD binding sequence had beendeleted (hereinafter referred to as the variant soybean PPO). Theoligonucleotides were prepared with a DNA synthesizer (PE AppliedBiosystems; Model 394 DNA/RNA synthesizer) and purified with anoligonucleotide purification cartridge (PE Applied Biosystems; OPCcartridge). The PCR was carried out by repeating a cycle for maintainingat 94° C. for 1 minute, at 55° C. for 2 minutes and the 72° C. for 3minutes 30 times. The amplified DNA fragments were digested withrestriction enzymes NcoI and SalI, and introduced between NcoIrestriction site and SalI restriction site of plasmid pTV118N(manufactured by Takara Shuzo Co., Ltd.) to construct plasmid pTVGMP(FIG. 8).

The plasmid pTVGMP was introduced into E. coli PPO gene deficient mutantBT3 strain according to the method described in Hanahan, D. J., Mol.Biol., 166; p 557 (1983). When the resultant E. coli were cultured inYPT medium containing 100 μg/ml ampicillin and 10 μg/ml kanamycin, nogrowth complemented clone was obtained.

EXAMPLE 12

Test for Effect of Giving Resistance to Herbicidal Compounds of VariantSoybean PPO

Plasmids pTVGMP and pTV118N prepared in Example 11 were introduced intoE. coli BT3/pACYCSP strain prepared in Example 3 respectively accordingto the method described in Flanahan, D. J., Mol. Biol., 166; p 557(1983). E. coli BT3/pACYCSP+pTVGMP strain bearing plasmids pACYCSP andpTVGMP, and E. coli BT3/pACYCSP+pTV118N strain bearing plasmids pACYCSPand pTV118N were obtained by culturing the above strains in YPT mediumcontaining 100 μg/ml ampicillin, 15 μg/ml chloramphenicol and 10 μg/mlkanamycin.

These E. coli strains were inoculated into YPT medium containing 10 or 1ppm of PPO inhibitory-type herbicidal compound represented by Structure8, 100 (ig/ml ampicillin, 15 μg/ml chloramphenicol, 10 μg/ml kanamycin,10 μg/ml hemin and 50 μg/ml aminolevulinic acid, cultured under darkconditions or light conditions according to the same manner as inExample 2. Then, 18 hours after initiation of culture, the absorbance ofliquid culture medium was measured at 600 nm. By taking the absorbanceof the medium without the herbicidal compound as 1, the relative valueof the absorbance of the medium containing the herbicidal compound wascalculated. The results are shown in Table 7.

TABLE 7 Relative absorbance Concentration of test Culture compound E.coli strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP + pTVGMP in thelight 0.33 0.85 1.0 BT3/pACYCSP + pTVGMP in the dark 0.91 0.94 1.0BT3/pACYCSP + pTV118N in the light 0.05 0.09 1.0 BT3/pACYCSP + pTV118Nin the dark 0.89 0.91 1.0

EXAMPLE 13

Introduction of the Gene Encoding Variant Soybean PPO into Tobacco

A plasmid for introducing the gene encoding the variant soybean PPO intoa plant by Agrobacterium infection method was constructed. PCR wascarried out by using the plasmid pSPPO-P prepared in Example 3 as atemplate, an oligonucleotide primer composed of the nucleotide sequenceof SEQ ID NO: 13 and an oligonucleotide primer composed of thenucleotide sequence of SEQ ID NO: 14 to amplify the DNA fragmentcontaining the gene encoding the variant soybean PPO. Then, plasmidpBI121K prepared in Example 9 was digested with the restriction enzymesKpnI and SacI to remove β-glucuronidase gene, and the DNA fragment whichwas obtained by digesting the DNA fragment containing the above geneencoding the variant soybean PPO with restriction enzymes KpnI and SacIwas inserted into this portion to prepare plasmid pBIGMP (FIG. 9) inwhich the gene was joined downstream from 35S promoter.

The plasmid pBIGMP was introduced into Agrobacterium tumefaciensLBA4404. The resultant transformants were cultured in a mediumcontaining 300 μg/ml streptomycin, 100 μg/ml rifampicin and 25 μg/mlkanamycin, followed by selecting the desired transformants to isolateAgrobacterium strain bearing pBIGMP.

Leaf pieces of tobacco cultured sterilely were infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco into which the gene encoding the variant soybean PPO wasintroduced was obtained.

EXAMPLE 14

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Gene Encoding Variant Soybean PPO

The level of resistance to PPO inhibitory type herbicidal compoundrepresented by Structure 8 was confirmed quantitatively by testingtobacco into which the gene encoding the variant soybean PPO prepared inExample 13 was introduced according to the same manner as in Example 6.The results obtained from 4 clones (GMP 1-4) of tobacco introduced withthe gene encoding the variant soybean PPO and control recombinanttobacco are shown in Table 8. In the table, the resistant level toherbicidal compound is represented by percentage of the totalchlorophyll content of leaf pieces treated with the herbicidal compoundto that of untreated leaf pieces.

TABLE 8 Total chlorophyll content (mg/g-fresh weight) ResistantRecombinant untreated- level to test tobacco leaf treated-leaf compound(%) control 3.49 0.35 10.0 GMP-1 1.89 2.55 135 GMP-2 0.89 0.96 108 GMP-31.50 1.49 99.3 GMP-4 2.91 2.34 80.4

EXAMPLE 15

Isolation of PPO Gene of Chlamydomonas Chlamydomonas reinhardtii CC407strain was obtained from Chlamydomonas Genetics Center (address: DCMBGroup, Department of Botany, Box 91000, Duke University, Durham, N.C.27708-1000, USA), cultured under 200 μE/m²/s photosynthesis active lightfor 5 days in TAP liquid culture medium (E. H. Harris, The ChlamydomonasSourcebook, Academic Press, San Diego, 1989, p 576-577) containing 7 mMNH₄Cl, 0.4 mM MgSO₄. 7H₂O, 0.34 mM CaCl₂.2H₂O, 25 mM potassiumphosphate, 0.5 mM Tris (pH 7.5), 1 ml/liter Hatner miner element and 1ml/liter glacial acetic acid to obtain 200 ml (1.0×106 cells/ml) liquidculture medium containing early stationary growth phase cells.

Total RNAs were prepared from these cells by using ISOGEN (manufacturedby Nippon Gene) according to the manual attached thereto. Also,poly(A)RNA was fractionated using BioMag mRNA Purification Kit(manufactured by Perceptive Bio System) according to the manual attachedthereto. cDNA was synthesized from the resultant poly(A)RNA by usingMarathon cDNA Amplification Kit (manufactured by Clontech) according tothe manual attached thereto and the cDNA was used as a template for PCR.

As PCR primers, an oligonucleotide composed of the nucleotide sequenceof SEQ ID NO: 15 and an oligonucleotide composed of the nucleotidesequence of SEQ ID NO: 16 were prepared. The oligonucleotides wereprepared with a DNA synthesizer (PE Applied Biosystems; Model 394DNA/RNA synthesizer) and purified with an oligonucleotide purificationcartridge (PE Applied Biosystems; OPC cartridge).

PCR was carried out by preparing a reaction liquid using Advantage cDNAPCR kit (manufactured by Clontech) according to the manual attachedthereto, and then, after maintaining at 94?C for 1 minute and then at65° C. for 5 minutes, repeating a cycle for maintaining at 94° C. for 15seconds and the 65° C. for 5 minutes 29 times. After the PCR, theamplified DNA fragments were purified by filtering the reaction liquidwith MicroSpin S-400HR (manufactured by Pharmacia Biotech), and the DNAfragment was cloned into plasmid pCR2.1 by using TA Cloning Kit(manufactured by Invitrogen) according to the manual attached thereto toconstruct plasmid pCPPO.

The nucleotide sequence of DNA fragment contained in the resultantplasmid pCPPO was determined by using Dye terminator cycle sequencingkit (manufactured by PE applied Biosystems) and DNA sequencer 373S(manufactured by PE applied Biosystems). As a result, the nucleotidesequence of SEQ ID NO: 17 was revealed, thereby confirming that plasmidpCPPO contained the full length PPO cDNA of Chlamydomonas reinhardtii.

EXAMPLE 16

Isolation of Gene Encoding Variant Protein of Chlamydomonas reinhardtiiPPO Having No Capability of Oxidizing Protoporphyrinogen IX and SpecificAffinity for Protoporphyrinogen IX

PCR was carried out by using plasmid pCPPO prepared in Example 15 as atemplate, and an oligonucleotide composed of the nucleotide sequence ofSEQ ID NO: 19 and an oligonucleotide composed of the nucleotide SEQ IDNO: 20 as primers to amplify the DNA fragment encoding Chlamydomonasreinhardtii PPO whose chloroplast transit signal and FAD bindingsequence had been deleted (hereinafter referred to as the variantChlamydomonas reinhardtii PPO). The oligonucleotides were prepared witha DNA synthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer)and purified with an oligonucleotide purification cartridge (PE AppliedBiosystems; OPC cartridge). The PCR was carried out by repeating a cyclefor maintaining at 94° C. for 1 minute, at 55° C. for 2 minutes and thenat 72° C. for 3 minutes 30 times. The amplified DNA fragment wasdigested with restriction enzymes BamHI and SacI, and inserted betweenBamHI restriction site and SacI restriction site of plasmid pTV119N(manufactured by Takara Shuzo Co., Ltd.) to construct plasmid pTVCRP(FIG. 10).

The plasmid pTVCRP was introduced into E. coli PPO gene deficient mutantBT3 strain according to the method described in Hanahan, D. J., Mol.Biol., 166; p 557 (1983). When the resultant E. coli were cultured inYPT medium containing 100 μg/ml ampicillin and 10 μg/ml kanamycin, nogrowth complemented clone was obtained.

EXAMPLE 17

Test of Variant Modified Chlamydomonas reinhardtii PPO for Capability ofGiving Resistance to Herbicidal Compounds

Plasmids pTVCRP and pTV118N prepared in Example 16 were introduced intoE. coli BT3/pACYCSP strain prepared in Example 3 respectively accordingto the method described in Hanahan, D. J., Mol. Biol., 166; p 557(1983). E. coli BT3/pACYCSP+pTVCRP strain bearing plasmids pACYCSP andpTVCRP, and E. coli BT3/pACYCSP+pTV118N strain bearing plasmids pACYCSPand pTV118N were obtained by culturing the above strains in YPT mediumcontaining 100 μg/ml ampicillin, 15 μg/ml chloramphenicol and 10 μg/mlkanamycin.

These E. coli strains were inoculated into YPT medium containing 10 or 1ppm of the PPO inhibitory-type herbicidal compound represented byStructure 8, 100 μg/ml ampicillin. 15 μg/ml chloramphenicol, 10 μg/mlkanamycin, 10 μg/ml hemin and 50 μg/ml aminolevulinic acid, culturedunder dark conditions or light conditions in the same manner as inExample 2. Then, 18 hours after initiation of culture, the absorbance ofliquid culture medium was measured at 600 nm. By taking the absorbanceof the medium containing no herbicidal compound as 1, the relative valueof the absorbance of the medium containing the herbicidal compound wascalculated. The results are shown in Table 9.

TABLE 9 Relative absorbance Concentration of test Culture compound E.coli strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP + pTVCRP in thelight 0.23 0.42 1.0 BT3/pACYCSP + pTVCRP in the dark 0.81 0.82 1.0BT3/pACYCSP + pTV118N in the light 0.12 0.24 1.0 BT3/pACYCSP + pTV118Nin the dark 0.80 0.91 1.0

EXAMPLE 18

Introduction of Gene Encoding Variant Chlamydomonas reinhardtii PPO intoTobacco

A plasmid for introducing the gene encoding the variant Chlamydomonasreinhardtii PPO into a plant by Agrobacterium infection method wasconstructed. The DNA fragment containing the gene encoding the variantChlamydomonas reinhardtii PPO was prepared by digesting plasmid pTVCRPprepared in Example 16 with restriction enzymes BamHI and ScaI. Binaryvector pBI121 (manufactured by Clontech) was digested with restrictionenzymes BamHI and ScaI to remove β-glucuronidase gene and the above geneencoding the variant Chlamydomonas reinhardtii PPO was inserted intothis portion to prepare plasmid pBICRP (FIG. 11).

The plasmid pBICRP was introduced into Agrobacterium tumefaciensLBA4404. The resultant transformants were cultured in a mediumcontaining 300 μg/ml streptomycin, 100 μg/ml rifampicin and 25 μg/mlkanamycin, followed by selecting the desired transformants to isolateAgrobacterium strain bearing pBICRP.

Leaf pieces of tobacco cultured sterilely were infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco into which the gene encoding the variant Chlamydomonasreinhardtii PPO was introduced was obtained.

EXAMPLE 19

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Gene Encoding Variant Chlamydomonas reinhardtii PPO

The level of resistance to the PPO-inhibitory type herbicidal compoundrepresented by Structure 8 was confirmed quantitatively by testingtobacco into which the gene encoding the variant Chlamydomonasreinhardtii PPO prepared in Example 18 was introduced according to thesame manner as in Example 6. The results obtained from 4 clones (CRP1-4) of tobacco into which the gene encoding the variant Chlamydomonasreinhardtii PPO was introduced and control recombinant tobacco is shownin Table 10. In the table, the resistant levels to the herbicidalcompound are represented by percentages of the total chlorophyll contentof leaf pieces treated with the herbicidal compound to that of untreatedleaf pieces.

TABLE 10 Total chlorophyll content (mg/g-fresh weight) ResistantRecombinant untreated- level to test tobacco leaf treated-leaf compound(%) control 2.28 0.42 18.4 CRP-1 1.27 1.54 121 CRP-2 1.50 1.67 111 CRP-31.10 1.11 101 CRP-4 1.58 1.57 99.4

EXAMPLE 20

Test of Variant Protein of Barley Ferrochelatase Having Affinity forProtoporphyrin IX Specifically for Capability of Giving Resistance toHerbicidal Compounds

A plasmid bearing barley ferrochelatase gene was prepared by the methoddescribed in Miyamoto. K. et al. Plant Physiol. 105; p 769 (1994). Theresultant plasmid was digested with restriction enzymes NspI and EcoRIto obtain the DNA fragment containing the gene encoding barleyferrochelatase whose signal sequence had been deleted (hereinafterreferred to as the variant barley ferrochelatase). This DNA fragment wasinserted between SphI restriction site and EcoRI restriction site ofplasmid pTV119N (manufactured by Takara Shuzo Co., Ltd.) to constructplasmid pTVHVF1 (FIG. 12).

The plasmids pTVHVF1 and pTV118N were introduced into E. coliBT3/pACYCSP strains prepared in Example 3 respectively according to themethod described in Hanahan, D. J., Mol. Biol., 166; p 557 (1983). E.coli BT3/pACYCSP+pTVHVF1 strain bearing plasmid pACYCSP and pTVHVF1, andE. coli BT3/pACYCSP+pTV118N strain bearing plasmid pACYCSP and pTV118Nwere obtained by culturing the above strains in YPT medium containing100 μg/ml ampicillin, 15 μg/ml chloramphenicol and 10 μg/ml kanamycin.

These E. coli strains were inoculated into YPT medium containing 10 or 1ppm of the PPO inhibitory-type herbicidal compound represented byStructure 8, 100 μg/ml ampicillin, 15 μg/ml chloramphenicol, 10 μg/mlkanamycin, 10 μg/ml hemin and 50 μg/ml aminolevulinic acid, culturedunder dark conditions or light conditions according to the same manneras in Example 2. Then, 18 hours after initiation of culture, theabsorbance of liquid culture medium was measured at 600 nm. By takingthe absorbance of the medium without the herbicidal compound as 1, therelative value of the absorbance of the medium containing the herbicidalcompound was calculated. The results are shown in Table 11.

TABLE 11 Relative absorbance Concentration of test Culture compound E.coli strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP + pTVHVF1 in thelight 0.39 0.94 1.0 BT3/pACYCSP + pTVHVF1 in the dark 0.94 0.96 1.0BT3/pACYCSP + pTV118N in the light 0.12 0.24 1.0 BT3/pACYCSP + pTV118Nin the dark 0.80 0.91 1.0

EXAMPLE 21

Introduction of the Gene Encoding Variant Barley Ferrochelatase intoTobacco

A plasmid for introducing the gene encoding barley ferrochelatase intotobacco by Agrobacterium infection method was constructed. The plasmidpTVHVF1 described in Example 20 was digested with restriction enzymeNcoI followed by blunting the DNA with DNA polymerase I by addingnucleotides to the double-stranded DNA gap. Then, the DNA wasdephosphorylated with alkaline phosphatase derived from calf intestineand cyclized by inserting phosphorylated BamHI linker (4610P,manufactured by Takara Shuzo Co., Ltd.) to construct plasmid pTVHVF2.Then, pTVHVF2 was digested with restriction enzyme EcoRI followed byblunting of the DNA with DNA polymerase I by adding nucleotides to thedouble-stranded DNA gap. Further, the DNA was dephosphorylated withalkaline phosphatase derived from calf intestine and cyclized byinserting phosphorylated SalI linker (4680P, manufactured by TakaraShuzo Co., Ltd.) to construct plasmid pTVHVF3. Plasmid pBI121KS preparedin Example 9 was digested with restriction enzymes BamHI and SalI toremove β-glucuronidase gene. The DNA fragment containing the geneencoding the variant barley ferrochelatase was prepared by digesting theabove pTVHVF3 with restriction enzymes BamHI and SalI. The resultant DNAfragment was inserted into plasmid pBI121KS with replacingβ-glucuronidase gene to prepare plasmid pBIHVF (FIG. 13) in whichvariant barley gene joined downstream from 35S promoter.

The plasmid pBIHVF was introduced into Agrobacterium tumefaciensLBA4404. The resultant transformants were cultured in a mediumcontaining 300 μg/ml streptomycin, 100 pg/ml rifampicin and 25 μg/mlkanamycin, followed by selecting the desired transformants to isolateAgrobacterium strain bearing pBIHVF.

Leaf pieces of tobacco cultured sterilely were infected with saidAgrobacterium strain and, according to the same manner as in Example 5,tobacco into which the gene encoding the variant barley ferrochelatasewas introduced was obtained.

EXAMPLE 22

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Gene Encoding Variant Barley Ferrochelatase

The level of resistance to the PPO inhibitory-type herbicidal compoundrepresented by Structure 8 was confirmed quantitatively by testingtobacco into which the gene encoding the variant barley ferrochelataseprepared in Example 21 was introduced according to the same manner as inExample 6. The results obtained from 4 clones (HVF 1-4) of tobaccointroduced with the gene encoding the variant barley ferrochelatase andcontrol recombinant tobacco are shown in table 12. In the table, theresistant levels to the herbicidal compound are represented bypercentages of the total chlorophyll content of leaf pieces treated withherbicidal compound to that of untreated leaf pieces.

TABLE 12 Total chlorophyll content (mg/g-fresh weight) ResistantRecombinant untreated- level to test tobacco leaf treated-leaf compound(%) control 1.93 0.160 8.29 HVF-1 0.876 0.930 106 HVF-2 1.14 1.16 102HVF-3 1.06 1.04 98.1 HVF-4 1.48 1.42 95.9

EXAMPLE 23

Test of Variant Protein of Cucumber Ferrochelatase Having SpecificAffinity for Protoporphyrin IX for Capability of Giving Resistance toHerbicidal Compounds

PCR was carried out by using cucumber ferrochelatase cDNA clone isolatedby the method described in Miyamoto, K. et al. Plant Physiol., 105; p769 (1994) as a template, an oligonucleotide composed of the nucleotidesequence of SEQ ID NO: 21 and an oligonucleotide composed of thenucleotide sequence of SEQ ID NO: 22 as primers to amplify the DNAfragment encoding cucumber ferrochelatase whose signal sequence had beendeleted (hereinafter referred to as the variant cucumberferrochelatase). The oligonucleotides were prepared with a DNAsynthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer) andpurified with an oligonucleotides purification cartridge (PE AppliedBiosystems; OPC cartridge). The PCR was carried out by repeating a cyclefor maintaining at 94° C. for 1 minute, at 55° C. for 2 minutes and thenat 72° C. for 3 minutes 30 times. The amplified DNA fragments weredigested with restriction enzymes BamHI and SacI, and inserted betweenBamHI restriction site and SacI restriction site of plasmid pTV119N(manufactured by Takara Shuzo Co., Ltd.) to construct plasmid pTVCSF(FIG. 14).

The plasmids pTVCSF and pTV118N were introduced into E. coli BT3/pACYCSPstrain prepared in Example 3 respectively according to the methoddescribed in Hanahan, D. J., Mol. Biol., 166; p 557 (1983). E. coliBT3/pACYCSP+ pTVCSF strain bearing plasmid pACYCSP and pTVCSF, and E.coli BT3/pACYCSP+pTV118N strain bearing plasmid pACYCSP and pTV118N wereobtained by culturing the above strains in YPT medium containing 100μg/ml ampicillin, 15 μg/ml chloramphenicol and 10 μg/ml kanamycin.

These E. coli strains were inoculated into YPT medium containing 10 or 1ppm of the PPO inhibitory-type herbicidal compound represented byStructure 8, 100 μg/ml ampicillin, 15 μg/ml chloramphenicol, 10 μg/mlkanamycin, 10 μg/ml hemin and 50 μg/ml aminolevulinic acid, culturedunder dark conditions or light conditions according to the same manneras in Example 2. Then, 18 hours after initiation of culture, theabsorbance of liquid culture medium was measured at 600 nm. By takingthe absorbance of the medium without the herbicidal compound as 1, therelative value of the absorbance of the medium containing the herbicidalcompound was calculated. The results are shown in Table 13.

TABLE 13 Relative absorbance Concentration of test Culture compound E.coli strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP + pTVCSF in thelight 0.73 0.78 1.0 BT3/pACYCSP + pTVCSF in the dark 0.89 0.92 1.0BT3/pACYCSP + pTV118N in the light 0.06 0.08 1.0 BT3/pACYCSP + pTV118Nin the dark 0.81 0.91 1.0

EXAMPLE 24

Introduction of the Gene Encoding Variant Cucumber Ferrochelatase intoTobacco

A plasmid for introducing the gene encoding the modified cucumberferrochelatase into tobacco by Agrobacterium infection method wasconstructed. Plasmid pBI121 (manufactured by Clontech) was digested withrestriction enzymes BamHI and ScaI to remove β-glucuronidase gene. A DNAfragment containing the gene encoding the variant cucumberferrochelatase was prepared by digesting plasmid pTVCSF described inExample 23 with restriction enzymes BamHI and ScaI. The resultant DNAfragment was introduced into plasmid pBI121 with replacingβ-glucuronidase gene to prepare plasmid pBICSF (FIG. 15) in whichvariant cucumber ferrochelatase gene was joined downstream from 35Spromoter.

The plasmid pBICSF was introduced into Agrobacterium tumefaciensLBA4404. The resultant transformants were cultured in a mediumcontaining 300 μg/ml streptomycin, 100 μg/ml rifampicin and 25 μg/mlkanamycin, followed by selecting the desired transformants to isolateAgrobacterium strain bearing pBICSF.

Leaf pieces of tobacco cultured sterilely were infected with saidAgrobacterium strain to obtain tobacco introduced with the gene encodingthe modified cucumber ferrochelatase according to the same manner as inExample 5.

EXAMPLE 25

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Gene Encoding Variant Cucumber Ferrochelatase

The level of resistance to PPO inhibitory-type herbicidal compounds isconfirmed quantitatively by testing tobacco introduced with the geneencoding the modified cucumber ferrochelatase prepared in Example 24according to the same manner as in Example 6.

EXAMPLE 26

Isolation of E. coli Coproporphyrinogen III Oxidase (hemF)

Gene

Genomic DNA was prepared from E. coli LE392 strain using Kit ISOPLANTfor genome DNA preparation (manufactured by Nippon Gene). Anoligonucleotide primer composed of the nucleotide sequence of SEQ ID NO:23 and an oligonucleotide primer composed of the nucleotide sequence ofSEQ ID NO: 24 were synthesized according to nucleotide sequences of its5′ and 3′ regions of E. coli hemF gene registered in GenBank (AccessionX75413). The oligonucleotides were prepared with a DNA synthesizer (PEApplied Biosystems; Model 394 DNA/RNA synthesizer) and purified with anoligonucleotides purification cartridge (PE Applied Biosystems; OPCcartridge). PCR was carried out by using about 1 μg of E. coli LE392strain genomic DNA as a template and the above oligonucleotides (each 10pmol) as primers to amplify the DNA fragment containing E. coli hemFgene. The PCR was carried out by repeating a cycle for maintaining at96° C. for 1 minute, at 55° C. for 2 minutes and then at 72° C. for 3minutes 30 times.

EXAMPLE 27

Test of E. coli hemF Protein for Capability of Giving Resistance toHerbicidal Compounds

The DNA fragment containing hemF gene amplified by the method describedin Example 26 was digested with restriction enzymes FbaI and PstI, andinserted between BamHI restriction site and PstI restriction site ofcommercially available plasmid pUC118N (manufactured by Takara ShuzoCo., Ltd.) to construct plasmid pHEMF (FIG. 16).

The plasmid pHEMF and pTV118N were introduced into E. coli BT3/pACYCSPstrain prepared in Example 3 respectively according to the methoddescribed in Hanahan, D. J., Mol. Biol., 166; p 557 (1983). E. coliBT3/pACYCSP+ pHEMF strain bearing plasmid pACYCSP and pHEMF, and E. coliBT3/pACYCSP+pTV118N strain bearing plasmid pACYCSP and pTV118N wereobtained by culturing the above strains in YPT medium containing 100μg/ml ampicillin, 15 μg/ml chloramphenicol and 10 μg/ml kanamycin.

These E. coli strains were inoculated into YPT medium containing 10 or 1ppm of the PPO inhibitory-type herbicidal compound represented byStructure 8, 100 μg/ml ampicillin, 15 μg/ml chloramphenicol, 10 μg/mlkanamycin, 10 μg/ml hemin and 50 μg/ml aminolevulinic acid, culturedunder dark conditions or light conditions according to the same manneras in Example 2. Then, 18 hours after initiation of culture, theabsorbance of liquid culture medium was measured at 600 nm. By takingthe absorbance of the medium without the herbicidal compound as I, therelative value of the absorbance of the medium containing the herbicidalcompound was calculated. The results are shown in Table 14.

TABLE 14 Relative absorbance Concentration of test Culture compoundE..coli strain conditions 10 ppm 1 ppm 0 ppm BT3/pACYCSP + pHEMF in thelight 0.48 1.0 1.0 BT3/pACYCSP + pHEMF in the dark 0.94 0.95 1.0BT3/pACYCSP + pTV118N in the light 0.06 0.16 1.0 BT3/pACYCSP + pTV118Nin the dark 0.96 0.98 1.0

EXAMPLE 28

Introduction of E. coli hemF Gene into Tobacco

A plasmid for introducing E. coli hemF gene into a plant byAgrobacterium infection method was constructed. First, for obtaining E.coli hemF gene, an oligonucleotide primer composed of the nucleotidesequence of SEQ ID NO: 25 and an oligonucleotide primer composed of thenucleotide sequence of SEQ ID NO: 26 were synthesized with a DNAsynthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer) andpurified with an oligonucleotide purification cartridge (PE AppliedBiosystems; OPC cartridge). PCR was carried out by using theoligonucleotide primers according to the same manner as in Example 26 toamplify the DNA fragment containing E. coli hemF gene.

Plasmid pBI121 (manufactured by Clontech) was digested with restrictionenzymes BamHI and SacI to remove β-glucuronidase gene. The DNA fragmentcontaining the gene encoding the E. coli hemF gene was prepared bydigesting the above PCR-amplified DNA fragment with restriction enzymesBamHI and SacI. The resultant DNA fragment was introduced into plasmidpBI121 with replacing β-glucuronidase gene to prepare plasmid pBIHEMF(FIG. 17) in which E. coli hemF gene was joined downstream from 35Spromoter.

The plasmid pBIHEMF was introduced into Agrobacterium tumefaciensLBA4404. The resultant transformants were cultured in a mediumcontaining 300 μg/ml streptomycin, 100 μg/ml rifampicin and 25 μg/mlkanamycin, followed by selecting the desired transformants to isolateAgrobacterium strain bearing pBIHEMF.

Leaf pieces of tobacco cultured sterilely were infected with theAgrobacterium strain to obtain tobacco introduced with E. coli hemF geneaccording to the same manner as in Example 5.

EXAMPLE 29

Confirmation of Resistance to Herbicidal Compounds of Tobacco Introducedwith the E. coli hemF Gene

The level of resistance to the PPO inhibitory-type herbicidal compoundsis confirmed quantitatively by testing tobacco introduced with the E.coli hemF gene (prepared in Example 28) according to the same manner asin Example 6.

EXAMPLE 30

Binding Test of Porphyrin Compound-Binding Protein to Protoporphyrin IX

A phage library presenting a protein containing an amino acid sequencecomposed of 5 random amino acids and a phage clone displaying a proteincontaining an amino acid sequence HASYS or RASSIL (wherein H ishistidine, A is alanine, S is serine, Y is tyrosine, R is arginine and Lis leucine) which can specifically bind to porphyrin compound 5, 10, 15,20-tetrakis (N-methylpyridinium-4-yl)-21H,23H-phosphine (H₂TMpyP) wereprepared according to the method described in KITANO et al., NihonKagakukai (Chemical Society of Japan) 74th Spring Annual MeetingPre-Published Abstracts of Presentation II, p 1353, 4G511 (1998).

First, the phage library displaying a protein containing an amino acidsequence composed of 5 random amino acids was constructed. Mixedoligonucleotides composed of the nucleotide sequence of SEQ ID NO: 27and mixed oligonucleotides composed of the nucleotide sequence of SEQ IDNO: 28 were synthesized. The mixed oligonucleotides were synthesizedwith a DNA synthesizer (PE Applied Biosystems; Model 394 DNA/RNAsynthesizer) and purified with an oligonucleotide purification cartridge(PE Applied Biosystems; OPC cartridge). The above mixed oligonucleotides(each 50 pmol) were phosphorylated at 5′ end by treating with T4 DNAkinase respectively. They were mixed and, after heating at 70° C. for 10minutes, subjected to annealing by cooling slowly to room temperature atrate of 0.5° C./minute. Plasmid pCANTAB5E (manufactured by PharmaciaBiotech) was digested with restriction enzymes SfiI and NotI to removethe recombinant antibody gene ScFv. The above phosphorylated andannealed oligonucleotide pair was inserted into the portion of the aboverecombinant antibody gene ScFv to prepare a plasmid containing anucleotide sequence encoding a protein composed of a 5 random amino acidsequence upstream from a protein comprising an amino acid sequence ofM13 phage coat protein. The plasmid was introduced into E. coli TG-1strain according to the method described in Hanahan, D. J., Mol. Biol.166; p 557 (1983) and cultured in 2× YT medium (10 g/liter yeastextract, 15 g/liter tryptone and 5 g/liter NaCl, pH 7.2) containing 100μg/ml ampicillin to obtain recombinant E. coli TG-1 strain. Therecombinant E. coli TG-1 strain was inoculated into 2× YT mediumcontaining 100 μg/ml ampicillin and cultured with shaking at 37

<C. Then, 1 hour after initiation of culture, 6×1010 pfu helper-phageM13K07 (manufactured by Pharmacia Biotech) was inoculated to the medium,and culture was continued for additional 18 hours with shaking. Then,the liquid culture medium was centrifuged at 1,000×g for 20 minutes tocollect the phage library displaying a protein containing the amino acidsequence composed of 5 random amino acids.

For preparing the phage clone displaying a protein containing the aminoacid sequence HASYS (SEQ ID NO: 53), an oligonucleotide composed of thenucleotide sequence of SEQ ID NO: 29 and an oligonucleotide composed ofthe nucleotide sequence of SEQ ID NO: 30 were synthesized. And, forpreparing the phage clone displaying a protein containing the amino acidsequence RASSL (SEQ ID NO: 55) an oligonucleotide composed of thenucleotide sequence of SEQ ID NO: 31 and an oligonucleotide composed ofthe nucleotide sequence of SEQ ID NO: 32 were synthesized. Theseoligonucleotides were synthesized with a DNA synthesizer (PE AppliedBiosystems; Model 394 DNA/RNA synthesizer) and purified with anoligonucleotide purification cartridge (PE Applied Biosystems; OPCcartridge). The phage clone displaying the protein containing the aminoacid sequence HASYS or RASSL was obtained by the same operation as theabove that for obtaining the phage library displaying a proteincontaining the amino acid sequence composed of 5 random amino acids.

A phage suspension containing the phage clone displaying the proteincontaining the amino acid sequence HASYS, the phage clone displaying theprotein containing the amino acid sequence RASSL or the phage librarydisplaying the protein containing the amino acid sequence consisting of5 random amino acids (titer 105 pfu) was respectively spotted to nitrocellulose filter (manufactured by Schleicher & Schuell), and then thenitro cellulose filter was blocked by shaking it in PBT buffer (137 mMNaCl, 8.10 mM Na₂HPO₄, 2.68 mM KCl, 1.47 mM KH₂PO₄, 0.05% Tween 20, pH7.2) containing 1% bovine serum albumin. The nitro cellulose filter waswashed with PBT buffer and shaken for 18 hours in 2×SSC buffer (0.3 MNaCl, 0.03M sodium citric acid) containing 10fÊM protoporphyrin IX.Further, said nitro cellulose filter was washed with 2×SSC buffer,dried, and fluorescence derived from protoporphyrin IX was detectedunder ultraviolet light (365 nm).

The spots of the phage library did not show fluorescence, while thespots of both phage clones displaying the protein containing the aminoacid sequence HASYS and that containing the amino acid sequence RASSLshowed clear fluorescence.

EXAMPLE 31

Test of Protoporphyrin IX Binding Protein for Capability of GivingResistance to Herbicidal Compounds

First, a plasmid which could express the gene encoding the proteincontaining the amino acid sequence HASYS (SEQ ID NO: 53), or the aminoacid sequence RASSL (SEQ ID NO: 55) was prepared. For preparing theplasmid capable of expressing the gene encoding the protein composed ofthe amino acid sequence of SEQ ID NO: 51 (hereinafter referred to as theprotein MGHASYS), an oligonucleotide composed of the nucleotide sequenceof SEQ ID NO: 33 and an oligonucleotide composed of the nucleotidesequence of SEQ ID NO: 34 were synthesized. The oligonucleotides weresynthesized with a DNA synthesizer (PE Applied Biosystems; Model 394DNA/RNA synthesizer) and purified with an oligonucleotide purificationcartridge (PE Applied Biosystems; OPC cartridge). The aboveoligonucleotides (each 50 pmol) were phosphorylated at 5′ end bytreating with T4 DNA kinase, respectively. They were mixed and then,after heating for 10 minutes at 70° C., subjected to annealing bycooling slowly to room temperature at rate of 0.5° C./minute. PlasmidpTV118N was digested with restriction enzymes NcoI and EcoRI to removethe gene fragment consisting of 16 base pairs. Plasmid pHASYS capable ofexpressing the gene encoding protein MGHASYS was prepared by insertedthe above phosphorylated and annealed oligonucleotide pairs into theposition of the above 16 base pairs.

Then, for preparing the plasmid capable of expressing the gene encodingthe protein consisting of amino acid sequence of SEQ ID NO: 56(hereinafter referred to as protein MGRASSL), an oligonucleotidecomposed of the nucleotide sequence of SEQ ID NO: 35 and anoligonucleotide composed of the nucleotide sequence of SEQ ID NO: 36were synthesized. The oligonucleotides were synthesized with a DNAsynthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer) andpurified with an oligonucleotide purification cartridge (PE AppliedBiosystems; OPC cartridge). Plasmid pRASSL capable of expressing thegene encoding protein MGRASSL was prepared by the same procedure as thatfor plasmid pHASYS.

A plasmid capable of expressing the gene encoding the protein containingthe amino acid sequence YAGY or YAGF (wherein Y is tyrosine, A isalanine, G is glycine, F is phenylalanine) (Sugimoto, N., Nakano. S.,Chem. Lett, p 939, 1997) capable of binding to porphyrin compoundH2TMPyP was prepared. For preparing the plasmid capable of expressingthe gene encoding the protein consisting of the amino acid sequence ofSEQ ID NO: 58 (hereinafter referred to as protein MGYAGY), anoligonucleotide composed of the nucleotide sequence of SEQ ID NO: 37 andan oligonucleotide composed of the nucleotide sequence of SEQ ID NO: 38were synthesized. For preparing the plasmid capable of expressing thegene encoding the protein composed of the amino acid sequence of SEQ IDNO: 60 (hereinafter referred to as protein MGYAGF), an oligonucleotidecomposed of the nucleotide sequence of SEQ ID NO: 39 and anoligonucleotide composed of the nucleotide sequence of SEQ ID NO: 40were also synthesized. These oligonucleotides were synthesized with aDNA synthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer)and purified with an oligonucleotide purification cartridge (PE AppliedBiosystems; OPC cartridge). Plasmid pYAGY capable of expressing the geneencoding the protein MGYAGY and plasmid pYAGF capable of expressing thegene encoding protein MGYAGF were prepared by the same procedure as thatfor plasmid pHASYS.

The above plasmids pHASYS, pRASSL, pYAGY, pYAGF and pTV118N wereintroduced into E. coli BT3/pACYCSP strain prepared in Example 3respectively according to the method described in Hanahan, D. J., Mol.Biol., 166; p 557 (1983). E. coli BT3/pACYCSP+pHASYS strain bearingplasmid pACYCSP and pHASYS, E. coli BT3/pACYCSP+pRASSL strain bearingplasmid pACYCSP and pRASSL, E. coli BT/pACYCSP+pYAGY strain bearingplasmid pACYCSP and pYAGY, E. coli BT3/pACYCSP+pYAGF strain bearingplasmid pACYCSP and pYAGF and E. coli BT3/pACYCSP+pTV118N strain bearingplasmid pACYCSP and pTV118N were obtained by culturing the above strainsin YPT medium containing 100 fÊg/ml ampicillin, 15 μg/ml chloramphenicoland 10 μg/ml kanamycin.

These E. coli strains were inoculated into YPT medium containing 1 ppmof the PPO inhibitory-type herbicidal compound represented by Structure8, 100 μg/ml ampicillin, 15 μg/ml chloramphenicol, 10 μg/ml kanamycin,10 μg/ml hemin and 50 μg/ml aminolevulinic acid, cultured under darkconditions or light conditions according to the same manner as inExample 2. Then, 18 hours after initiation of culture, the absorbance ofliquid culture medium was measured at 600 nm. By taking the absorbanceof the medium without the herbicidal compound as 1, the relative valueof the absorbance of the medium containing the herbicidal compound wascalculated. The results are shown in Table 15.

TABLE 15 Relative absorbance Concentration of Culture test compound E.coli strain conditions 1 ppm 0 ppm BT3/pACYCSP + pHASYS in the light0.65 1.0 BT3/pACYCSP + pHASYS in the dark 0.96 1.0 BT3/pACYCSP + pRASSLin the light 0.59 1.0 BT3/pACYCSP + pRASSL in the dark 1.0 1.0BT3/pACYCSP + pYAGY in the light 0.48 1.0 BT3/pACYCSP + pYAGY in thedark 0.99 1.0 BT3/pACYCSP + pYAGF in the light 0.62 1.0 BT3/pACYCSP +pYAGF in the dark 0.96 1.0 BT3/pACYCSP + in the light 0.07 1.0 pTV118NBT3/pACYCSP + in the dark 0.93 1.0 pTV118N

Further, a plasmid capable of expressing a gene encoding a proteincontaining an amino acid sequence in which one unit of the amino acidsequences HASYS or RASSL were repeatedly joined. For preparing theplasmid capable of expressing the gene encoding the protein composed ofthe amino acid sequence of SEQ ID NO: 61 (hereinafter referred to asprotein MG(HASYS)4, (HASYS)n referred to as a sequence in which peptideHASYS was repeatedly joined to each other n times), an oligonucleotidecomposed of the nucleotide sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQID NO: 43 or SEQ ID NO: 44 was synthesized. These oligonucleotides weresynthesized with a DNA synthesizer (PE Applied Biosystems; Model 394DNA/RNA synthesizer) and purified with an oligonucleotide purificationcartridge (PE Applied Biosystems: OPC cartridge). First, theoligonucleotide composed of the nucleotide sequence of SEQ ID NO. 42 andthe oligonucleotide composed of the nucleotide sequence of SEQ ID NO: 43were phosphorylated respectively at 5′ end by treating with T4 DNAkinase. Thereafter, the oligonucleotide composed of the nucleotidesequence of SEQ ID NO: 41 and the oligonucleotide composed of thephosphorylated nucleotide sequence of SEQ ID NO. 42 or theoligonucleotide composed of the phosphorylated nucleotide sequence ofSEQ ID NO: 43 and the oligonucleotide composed of the nucleotidesequence of SEQ ID NO: 44 were mixed (each 300 pmol), and after heatingfor 5 minutes at 70° C., annealed by cooling slowly to room temperatureat rate of 0.5° C./minute. The above two annealed oligonucleotide pairswere mixed and ligated with T4 DNA ligase, then the resultant DNAfragment was phosphorylated with T4 DNA kinase at 5′ end. On the otherhand, vector pTV118N was digested with restriction enzymes NcoI andEcoRI to remove a DNA fragment of 16 base pairs and the abovephosphorylated DNA fragment was inserted into this portion to obtainplasmid pHASYS4 expressing the gene encoding protein MG(HASYS)4.

Further, for preparing the plasmid capable of expressing the geneencoding the protein composed of the amino acid sequence of SEQ ID NO:62 (hereinafter referred to as protein MG(HASYS)8), an oligonucleotidecomposed of the nucleotide sequence of SEQ ID NO: 45and anoligonucleotide composed of the nucleotide sequence of SEQ ID NO: 46were synthesized. These oligonucleotides were synthesized with a DNAsynthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer) andpurified with an oligonucleotide purification cartridge (PE AppliedBiosystems; OPC cartridge). First, the above oligonucleotides werephosphorylated at 5′ and by treating with T4 DNA kinase. Thereafter, anoligonucleotide composed of the nucleotide sequence of SEQ ID NO: 41 andan oligonucleotide composed of the phosphorylated nucleotide sequence ofSEQ ID NO: 42 were mixed (each 300 pmol), an oligonucleotide composed ofthe phosphorylated nucleotide sequence of SEQ ID NO: 43 and anoligonucleotide composed of the nucleotide sequence of SEQ ID NO: 44were mixed (each 300 pmol), and further, an oligonucleotide composed ofthe phosphorylated nucleotide sequence of SEQ ID NO: 45 and anoligonucleotide composed of the phosphorylated nucleotide sequence ofSEQ ID NO: 46 were mixed (each 600 pmol). These three mixtures wereheated for 5 minutes at 70° C., and annealed by cooling slowly to roomtemperature at rate of 0.5° C./minute, respectively. The above threeannealed oligonucleotide pairs were mixed, and ligated with T4 DNAligase, and then the resultant DNA fragment was phosphorylated with T4DNA kinase at 5′ end. Plasmid pHASYS8 capable of expressing proteinMG(HAS YS)8 were prepared in the same manner as that for the aboveplasmid pHASYS4.

Then, for preparing a plasmid capable of expressing the gene encodingthe protein composed of the amino acid sequence of SEQ ID NO: 63(hereinafter referred to as protein MG(RASSL)4, (RASSL)n referred to asa sequence in which peptide RASSL was repeatedly joined to each other ntimes), an oligonucleotide composed of the nucleotide sequence of SEQ IDNO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50 were synthesized.Also, for preparing a plasmid capable of expressing the gene encodingthe protein composed of the amino acid sequence of SEQ ID NO: 64(hereinafter referred to as protein MG(RASSL)8), an oligonucleotidecomposed of the nucleotide sequence of SEQ ID NO: 51 and anoligonucleotide composed of the nucleotide sequence of SEQ ID No: 52were synthesized. These oligonucleotides were synthesized with a DNAsynthesizer (PE Applied Biosystems; Model 394 DNA/RNA synthesizer) andpurified with an oligonucleotide purification cartridge (PE AppliedBiosystems; OPC cartridge).

Plasmid pRASSL4 capable of expressing protein MG(RASSL)4 were preparedaccording to the same manner as that for the above plasmid pHASYS4.Plasmid pRASSL8 capable of expressing protein MG(RASSL)8 were alsoprepared according to the same manner as that for the above plasmidpHASYS8.

The above plasmids pHASYS4, pHASYS8, pRASSL4, pRASSL8 and pTV118N wereintroduced into E. coli BT3/pACYCSP strain prepared in Example 3respectively according to the method described in Hanahan, D. J., Mol.Biol., 166; p 557 (1983). E. coli BT3/pACYCSP+pHASYS4 strain bearingplasmid pACYCSP and pHASYS4, E. coli BT3/pACYCSP+pHASYS8 strain bearingplasmid pACYCSP and pHASYS8, E. coli BT3/pACYCSP+pRASSL4 strain bearingplasmid pACYCSP and pRASSL4, E. coli BT3/pACYCSP+pRASSL8 strain bearingplasmid pACYCSP and pRASSL8 and E. coli BT3/pACYCSP+pTV118N strainbearing plasmid pACYCSP and pTV118N were obtained by culturing the abovestrains in YPT medium containing 100 μg/ml ampicillin, 15 μg/mlchloramphenicol and 10 μg/ml kanamycin.

These E. coli strains were inoculated into YPT medium containing 1 ppmof the PPO inhibitory-type herbicidal compound represented by Structure8, 100 μg/ml ampicillin, 15 μg/ml chloramphenicol, 10 μg/ml kanamycin,10 μg/ml hemin and 50 μg/ml aminolevulinic acid, cultured under darkconditions or light conditions according to the same manner as inExample 2. Then, 18 hours after initiation of culture, the absorbance ofthe liquid culture medium was measured at 600 nm. By taking theabsorbance of the culture medium without the herbicidal compound as 1,the relative value of the absorbance of the culture medium containingthe herbicidal compound was calculated. The results are shown in Table16.

TABLE 16 Relative absorbance Concentration of Culture test compound E.coli strain condition 1 ppm 0 ppm BT3/pACYCSP + pHASYS4 in the light0.91 1.0 BT3/pACYCSP + pHASYS4 in the dark 1.0 1.0 BT3/pACYCSP + pHASYS8in the light 0.57 1.0 BT3/pACYCSP + pHASYS8 in the dark 1.0 1.0BT3/pACYCSP + pRASSL4 in the light 1.1 1.0 BT3/pACYCSP + pRASSL4 in thedark 0.98 1.0 BT3/pACYCSP + pRASSL8 in the light 0.79 1.0 BT3/pACYCSP +pRASSL8 in the dark 1.0 1.0 BT3/pACYCSP + pTV118N in the light 0.15 1.0BT3/pACYCSP + pTV118N in the dark 0.81 1.0

EXAMPLE 32

Introduction of the Gene Encoding Protoporphyrin IX Binding Peptide intoTobacco

A plasmid for introducing the gene encoding the protoporphyrin IXbinding peptide into tobacco by Agrobacterium method was constructed.The plasmid pHASYS8 prepared in Example 31 was digested with restrictionenzyme NcoI followed by blunting the DNA with DNA polymerase I withaddition of nucleotides to the double-stranded DNA gap. Then, the DNAwas dephosphorylated with alkaline phosphatase derived from calfintestine and cyclized by inserting phosphorylated BamHI linker (4610P,manufactured by Takara Syuzo Co., Ltd.) to construct plasmid pHASYS8B.Plasmid pBI121 (manufactured by Clonetech) was digested with restrictionenzymes BamHI and ScaI to remove β-glucuronidase gene. On the otherhand, plasmid pHASYS8B was digested with restriction enzymes BamHI andScaI to prepare the DNA fragment containing the gene encoding proteinMG(HASYS)8, the resultant DNA fragment was inserted into plasmid pBI121with replacing β-glucuronidase gene to prepare plasmid pBIHASYS8 (FIG.18) in which the gene encoding protoporphyrin IX binding proteinMG(HASYS)8 was joined downstream from 35S promoter.

A plasmid for introducing the gene encoding the protoporphyrin IXbinding peptide MG(RASSL)8 into a plant by Agrobacterium infectionmethod was constructed. Plasmid pBIRASSL8 (FIG. 19) in which the geneencoding protoporphyrin IX binding protein MG(RASSL)8 was joineddownstream from 35S promoter was prepared from pRASSL8 according to thesame procedure as that for pBIHASYS8.

The above plasmid pBIHASYS8 and pBIRASSL8 were introduced intoAgrobacterium tumefaciens LBA4404 respectively. The resultanttransformants were cultured in a medium containing 300 μg/mlstreptomycin, 100 μg/ml rifampicin and 25 μg/ml kanamycin, followed byselecting the desired transformants to isolate Agrobacterium strainsbearing pBIHASYS8 and pBIRASSL8, respectively.

Leaf pieces of tobacco cultured sterilely are infected with saidAgrobacterium strains to obtain tobacco introduced with the geneencoding protoporphyrin IX binding protein MG(HASYS)8, and the tobaccointroduced with the gene encoding protoporphyrin IX binding proteinMG(RASSL)8 in the same manner as in Example 5.

EXAMPLE 33

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Gene Encoding the Protoporphyrin IX Binding Peptide

The level of resistance to herbicidal compounds is confirmedquantitatively by testing tobacco introduced with the gene encoding theprotoporphyrin IX binding peptide prepared in Example 32 according tothe same manner as in Example 14.

EXAMPLE 34

Isolation of PPO Gene of Arabidopsis thaliana Having HerbicidalCompound-Resistant Mutation

A plasmid containing PPO gene of Arabidopsis thaliana obtained by themethod described by Narita, S. et al., Gene, 182; p 169 (1996) wasdigested with the restriction enzyme NcoI, and nucleotides were added tothe gap of the double-stranded DNA by using DNA Polymerase I to bluntthe end of the DNA. Then, the 5′-end of the DNA was dephosphorylatedwith an alkaline phosphatase derived from calf small intestine, followedby insertion of a phosphorylated BamHI linker (4810P manufactured byTakara Shuzo Co., Ltd.) therein and cyclization to construct plasmidpAGE17B. The plasmid pAGE17B was digested with BamHI and ScaI to obtaina gene fragment containing PPO gene of Arabidopsis thaliana. Thefragment was inserted between BamHI and ScaI of a commercially availablevector, pKF19k for site-directed mutagenesis (manufactured by TakaraShuzo Co., Ltd.), to construct plasmid pKFATP.

Then, for conversion of the 220th alanine into valine, a PPOinhibitory-type herbicidal compound-resistant mutation in PPO protein ofArabidopsis thaliana disclosed in WO 9534659, base substitution(substitution of “T” for the 659 base “C”) of DNA was introduced intothe above PPO gene of Arabidopsis thaliana. First, an oligonucleotideprimer for mutagenesis represented by SEQ ID NO: 65 was synthesized. Theoligonucleotide primer was synthesized with a DNA synthesizer (PEApplied Biosystems: Model 394 DNA/RNA Synthesizer) and purified with anoligonucleotide purification cartridge (PE Applied Biosystems: OPCCartridge). The 5′-end of the oligonucleotide primer was phosphorylatedwith T4 DNA kinase. According to the manual attached to a commerciallyavailable site-directed mutagenesis kit, Mutan-Super Express Km(manufactured by Takara Shuzo Co., Ltd.), a reaction mixture containing10 ng of the above plasmid pKFATP as template DNA, 5 pmol of theattached selection primer, 5 pmol of the above phosphorylatedoligonucleotide primer for mutagenesis and the like was prepared and PCRwas carried out. The PCR was carried by repeating a cycle formaintaining at 94° C. for 1 minute, at 55° C. for 1 minute and then at72° C. for 3 minutes 30 times. The resultant reaction mixture waspurified by ethanol precipitation and the precipitate was dissolved in 5μl of sterilized-distilled water. According to the attached manual, its2 μl portion was used for introduction into a commercially available E.coli competent cell, MV1184 (manufactured by Takara Shuzo Co., Ltd.),and plated on LB agar culture medium (1% tryptone, 0.5% yeast extract,0.5% NaCl, 1% agar) containing 50 μg/ml of kanamycin. After incubationat 37° C., the resultant clone was cultured in LB liquid mediumcontaining 50 μg/ml of kanamycin to prepare plasmid DNA. Theintroduction of the desired herbicidal compound-resistant mutation A220Vwas confirmed by analyzing the nucleotide sequence of the PPO gene ofArabidopsis thaliana having herbicidal compound-resistant mutationcontained in the resultant plasmid pKFATP1.

EXAMPLE 35

Introduction of PPO Gene of Arabidopsis thaliana Having HerbicidalCompound-Resistant Mutation Into Tobacco

A plasmid was constructed for introducing the PPO gene of Arabidopsisthaliana having herbicidal compound-resistant mutation (hereinafterreferred to as Arabidopsis thaliana PPO(A220V) gene) into a plant byAgrobacterium infection method. Binary vector pBI121 (manufactured byClontech) was digested with the restriction enzymes BamHI and SacI toremove β-glucuronidase gene. On the other hand, the plasmid pKFATP1described in Example 34 was digested with restriction enzymes BamHI andSacI to prepare a DNA fragment containing Arabidopsis thalianaPPO(A220V) gene. Instead of the above β-glucuronidase gene, theresultant DNA fragment was inserted in the binary vector pBI121 toconstruct the plasmid pNATP (FIG. 20).

The plasmid pNATP was introduced into Agrobacterium tumefaciens LBA4404and this was cultured in LB culture medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pNATP.

Tobacco leaf pieces cultured sterilely were infected with theAgrobacterium strain and, according to the same manner as described inExample 5, tobacco bearing the introduced Arabidopsis thalianaPPO(A220V) gene was obtained.

EXAMPLE 36

Production of Recombinant Tobacco Having Arabidopsis thaliana PPO(A220V)Gene and Gene Encoding Variant Tobacco Chelatase Subunit

A plasmid was constructed for introducing both Arabidopsis thalianaPPO(A220V) gene and gene encoding a variant tobacco chelatase subunitinto a plant by Agrobacterium infection method. First, oligonucleotideprimers composed of the nucleotide sequence represented by SEQ ID NO: 66and the nucleotide sequence represented by SEQ ID NO: 67, respectively,were synthesized. The oligonucleotide primers were synthesized by a DNAsynthesizer (PE Applied Biosystems: Model 394 DNA/RNA Synthesizer) andpurified with an oligonucleotide purification cartridge (PE AppliedBiosystems: OPC Cartridge). PCR was carried out by using the primers andthe plasmid pKFATP1 constructed in Example 34 as template DNA to preparea DNA fragment containing Arabidopsis thaliana PPO(A220V) gene. The PCRwas carried out by repeating a cycle for maintaining at 94° C. for 1minute, 55° C. for 2 minutes and then 72° C. for 3 minutes 30 times. Theamplified DNA fragment was digested by the restriction enzymes HindIIIand SalI to obtain a DNA fragment containing Arabidopsis thalianaPPO(A220V) gene. The fragment was inserted between HindIII and SalIrestriction sites of a commercially available vector, pUC19, toconstruct plasmid pAP.

On the other hand, the plasmid pNG01 (FIG. 29) described in Shiota, N.et al., Plant Physiol., 106; p 17 (1994) was digested with therestriction enzyme Hindi 11 and nucleotides were added to the gap of thedouble-stranded DNA with DNA Polymerase I to blunt the end of the DNA,followed by self-cyclization to construct the plasmid pNG04 (FIG. 30).The plasmid pNG04 was digested with the restriction enzyme XbaI toisolate a DNA fragment of about 1.1 kb composed of the terminator of anopaline synthase and 35S promoter. The DNA fragment was inserted intothe XbaI restriction site of the above plasmid pAP. For selectingplasmid pAPNS wherein the terminator of the nopaline synthase wasligated to the downstream of Arabidopsis thaliana PPO(A220V) gene,digestion with the restriction enzymes HindIII and PstI was carried outto select a clone producing a DNA fragment of about 2.0 kb composed ofArabidopsis thaliana PPO(A220V) gene and the terminator of the nopalinesynthase. Further, the plasmid pAPNS was digested with the restrictionenzyme HindIII and nucleotides were added to the gap of thedouble-stranded DNA with DNA polymerase I to blunt the end of the DNA.The 5′-end of the DNA was dephosphorylated with an alkaline phosphatasederived from calf small intestine and a phosphorylated KpnI linker(4668P manufactured by Takara Shuzo Co., Ltd.) was inserted therein,followed by cyclization to construct plasmid pAPNSK.

The plasmid pAPNSK was digested with the restriction enzymes KpnI andDraI to isolate a DNA fragment of about 2.8 kb composed of Arabidopsisthaliana PPO(A220V) gene, the terminator of the nopaline synthaselocated at the downstream of the gene and 35S promoter located at thedownstream of the terminator. The fragment was inserted in KpnIrestriction site of the plasmid pBITCHLH constructed in Example 9. Forselecting the plasmid pBIAPTCH (FIG. 21) wherein Arabidopsis thalianaPPO(A220V) gene and a gene encoding a variant tobacco chelatase subunitwere ligated to the downstream of 35S promoter, respectively, theresultant plasmid was digested with the restriction enzyme BamHI toselect a clone producing a DNA fragment of about 2.8 kb composed ofArabidopsis thaliana PPO(A220V) gene, the terminator of the nopalinesynthase and the 35S promoter.

The plasmid pBIAPTCH was introduced into Agrobacterium tumefaciensLBA4404 and this was cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml rifampicin and 25 μg/ml kanamycin, followed byselection of a transformant to isolate an Agrobacterium strain bearingpBIAPTCH.

Tobacco leaf pieces cultured sterilely were infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco bearing the introduced both Arabidopsis thaliana PPO(A220V) geneand gene encoding the variant tobacco chelatase subunit was obtained.

EXAMPLE 37

Confirmation of Resistance to Herbicidal Compound of Tobacco BearingIntroduced Arabidopsis thaliana PPO(A220V) Gene and Gene EncodingVariant Tobacco Chelatase Subunit

Leaves of the tobacco bearing the introduced both Arabidopsis thalianaPPO(A220V) gene and gene encoding the variant tobacco chelatase subunitproduced in Example 36, those bearing the introduced Arabidopsisthaliana PPO(A220V) gene produced in Example 35, and the controlrecombinant tobacco leaves produced in Example 5were collected, and eachleaf was divided into the right and left equivalent pieces along themain vein. One of the pieces was treated with an aqueous solutioncontaining 2.0 ppm PPO inhibitory-type herbicidal compound of Structure8, while the other piece was not treated with the compound. These leafpieces were placed on MS medium containing 0.8% agar and allowed tostand at room temperature for 7 days in a light place. Then, each leafpiece was ground in 5 ml of 80% aqueous acetone solution in a mortarwith a pestle to extract chlorophyll. The extract was diluted 10 timeswith 80% aqueous acetone solution and the absorbance was measured at 750nm, 663 nm and 645 nm to calculate the total chlorophyll contentaccording to the method described by Macknney G., J. Biol. Chem. (1941)140, p 315. The resistant level to the herbicidal compound tested wasrepresented by the percentage of the total chlorophyll content of theleave pieces treated with the herbicidal compound to that of untreatedleaf pieces. The resistant level of the control recombinant tobacco was2.88% and that of the tobacco bearing the introduced Arabidopsisthaliana PPO(A220V) gene was 12.2%. On the other hand, the resistantlevel of the tobacco bearing introduced both Arabidopsis thalianaPPO(A220V) gene and gene encoding the variant tobacco chelatase subunitwas 61.6%.

EXAMPLE 38

Isolation of Gene Encoding Chloroplast-Localized Type Ferrochelatase ofArabidopsis thaliana

Total RNAs were prepared from leaf tissues of Arabidopsis thalianaecotype WS by using RNeasy Plant Kit (manufactured by QIAGEN) accordingto the manual attached thereto. A DNA fragment containing achloroplast-localized type ferrochelatase gene of Arabidopsis thalianawas obtained by using RNA LA PCR Kit (AMV) Ver 1.1 (manufactured byTakara Shuzo Co., Ltd.) according to the manual attached thereto. First,1st strand cDNA was synthesized by using Arabidopsis thaliana total RNAsas a template and Oligo dT-Adaptor Primer contained in the above kit asthe primer with the reverse transcriptase contained in the above kit.Then, PCR was carried out by using the 1st strand cDNA as a template andLA Taq polymerase contained in the above kit to amplify a DNA fragmentcontaining the gene encoding the chloroplast-localized typeferrochelatase of Arabidopsis thaliana. In this PCR, the primers usedwere the oligonucleotide primer composed of the nucleotide sequence ofSEQ ID NO: 68 and the oligonucleotide primer composed of the nucleotidesequence of SEQ ID NO: 69. The oligonucleotides were synthesized by aDNA synthesizer (PE Applied Biosystems: Model 394 DNA/RNA Synthesizer)and purified with an oligonucleotide purification cartridge (PE AppliedBiosystems: OPC Cartridge). The PCR was earned by maintaining at 94° C.for 2 minutes and then repeating a cycle for maintaining at 94° C. for30 seconds, at 50° C. for 30 seconds and then at 72° C. for 7 minutes 30times. After the PCR, the DNA fragment amplified by the PCR was clonedinto the plasmid pCR2.1 by using TA Cloning Kit (manufactured byInvitrogen) according to the manual attached thereto. The resultantplasmid was digested with the restriction enzyme BamHI and analyzed byagarose gel electrophoresis. The plasmid from which 5.3 kb DNA fragmentwas detected was named pCRATF (FIG. 22). The plasmid has such astructure that the chloroplast-localized type ferrochelatase gene ofArabidopsis thaliana is inserted in a DNA strand complementary to thelac promoter. When the nucleotide sequence of the chloroplast-localizedtype ferrochelatase gene of Arabidopsis thaliana was analyzed, it agreedwith the nucleotide sequence of the chloroplast-localized typeferrochelatase gene of Arabidopsis thaliana described by Smith, A. G. etal., J. Biol, chem., 269; p 13405 (1994).

EXAMPLE 39

Introduction of Gene Encoding Chloroplast-localized Type Ferrochelataseof Arabidopsis thaliana

A plasmid was constructed for introducing a gene encodingchloroplast-localized type ferrochelatase of Arabidopsis thaliana into aplant by Agrobacterium infection method. First, the plasmid pCRATFconstructed in Example 38 was digested with the restriction enzymesBamHI and SacI to prepare a DNA fragment containingchloroplast-localized type ferrochelatase gene of Arabidopsis thaliana.Binary vector pBI121 (manufactured by Clontech) was digested with therestriction enzymes BamHI and SacI to remove β-glucuronidase gene and,instead of this gene, the above DNA fragment containing thechloroplast-localized type ferrochelatase gene of Arabidopsis thalianawas inserted therein to construct the plasmid pBIATF (FIG. 23) whereinthe ferrochelatase gene was ligated to the downstream of the 35Spromoter.

The plasmid pBIATF was inserted into Agrobacterium tumefaciens LBA4404and this was cultured in LB medium containing 300 μg/ml of streptomycin,100 μg/ml of rifampicin and 25 μg/ml of kanamycin, followed by selectionof a transformant to isolate an Agrobacterium strain bearing pBIATF.

Tobacco leaf pieces cultivated sterilely are infected with theAgrobacterium strain and tobacco bearing the introducedchloroplast-localized type ferrochelatase gene of Arabidopsis thalianais obtained according to the same manner as in Example 5.

EXAMPLE 40

Production of Recombinant Tobacco Bearing Both Arabidopsis thalianaPPO(A220V) Gene and Chloroplast-Localized Type Ferrochelatase Gene ofArabidopsis thaliana

A plasmid is constructed for introducing both Arabidopsis thalianaPPO(A220V) gene and chloroplast-localized type ferrochelatase gene ofArabidopsis thaliana into a plant by Agrobacterium infection method.First, the plasmid pAPNS constructed in Example 36 are digested with therestriction enzyme HindIII and nucleotides are added to the gap of thedouble-stranded DNA with DNA Polymerase I to blunt the end of the DNA.The 5′-end of the DNA is dephosphorylated with an alkaline phosphatasederived from calf small intestine and a phosphorylated BamHI linker(4610P manufactured by Takara Shuzo Co., Ltd.) is inserted therein,followed by cyclization to construct plasmid pAPNSB.

The plasmid pAPNSB is digested with the restriction enzymes BamHI andDraI to isolate a DNA fragment of about 2.8 kb composed of Arabidopsisthaliana PPO(A220V) gene, the terminator of nopaline synthase and 35Spromoter. The fragment is inserted in the BamHI restriction site of theplasmid pBIATF constructed in Example 39. For selecting the plasmidpBIAPATF (FIG. 24) wherein Arabidopsis thaliana PP(A220V) gene andchloroplast-localized type ferrochelatase gene of Arabidopsis thalianaare ligated to the downstream of 35S promoter, the resultant plasmid isdigested with the restriction enzymes NotI and ScaI to select a cloneproducing a DNA fragment of about 2.2 kb composed of the 35S promoterand the chloroplast-localized type ferrochelatase gene of Arabidopsisthaliana.

The plasmid pBIAPATF is introduced into Agrobacterium tumefaciensLBA44044 and this is cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBIAPATF.

Tobacco leaf pieces cultivated sterilely are infected with theAgrobacterium strain and tobacco bearing the introduced Arabidopsisthaliana PPO(A220V) gene and chloroplast-localized type ferrochelatasegene of Arabidopsis thaliana is obtained according to the same manner asin Example 5.

EXAMPLE 41

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Arabidopsis thaliana PPO(A220V) Gene andChloroplast-Localized Type Ferrochelatase Gene of Arabidopsis thaliana

The levels of resistance to herbicidal compounds are confirmedquantitatively by testing the tobacco bearing the introduced Arabidopsisthaliana PPO(A220V) gene and chloroplast-localized type ferrochelatasegene of Arabidopsis thaliana produced in Example 40 according to thesame manner as in Example 3.

EXAMPLE 42

Isolation of Soybean Coproporphyrinogen III Oxidase Gene

Total RNAs were prepared from leaf tissues of soybean (Glycine max cv.Jack) by using RNeasy Plant Kit (manufactured by QIAGEN) according tothe manual attached thereto. Further, a DNA fragment containing a geneencoding soybean coproporphyrinogen III oxidase (hereinafter referred toas the present soybean CPOX) was obtained by using RNA LA PCR Kit (AMV)Ver 1.1 (manufactured by Takara Shuzo Co., Ltd.) according to the manualattached thereto. First, 1st strand cDNA was synthesized by using thesoybean total RNAs as a template and Oligo dT-Adaptor Primer containedin the above kit as a primer with the reverse transcriptase contained inthe above kit. Then, PCR was carried out by using the 1st strand cDNA asa template and LA Taq polymerase contained in the above kit to amplify aDNA fragment containing the present soybean CPOX gene. In this PCR, anoligonucleotide primer composed of the nucleotide sequence representedby SEQ ID NO: 70 and an oligonucleotide primer composed of thenucleotide sequence represented by SEQ ID NO: 71 were used. Theseoligonucleotides were synthesized by using a DNA synthesizer (PE AppliedBiosystems: Model 394 DNA/RNA Synthesizer) and purified with anoligonucleotide purification cartridge (PE Applied Biosystems: OPCCartridge). The PCR was carried out by maintaining at 94° C. for 2minutes and then repeating a cycle for maintaining at 94° C. for 30seconds, at 50° C. for 30 seconds and then at 72° C. for 7 minutes 30times. After the PCR, the DNA fragment amplified by the PCR was clonedinto plasmid pCR2.1 by using TA Cloning Kit (manufactured by Invitrogen)according to the manual attached thereto. The resultant plasmid wasdigested with the restriction enzyme BamHI and analyzed by agarose gelelectrophoresis. The plasmid from which 1.2 kb DNA fragment was detectedwas named pCRSCPOX (FIG. 25). The plasmid pCRSCPOX has such a structurethat the present soybean CPOX gene is inserted into a DNA strandcomplementary to the lac promoter. When the nucleotide sequence of theDNA fragment in the plasmid was analyzed, it was confirmed to be thepresent soybean CPOX gene.

EXAMPLE 43

Introduction of Present Soybean CPOX Gene into Tobacco

A plasmid was constructed for introducing the present soybean CPOX geneinto a plant by Agrobacterium infection method. First, the plasmidpCRSCPOX constructed in Example 42 was digested with the restrictionenzymes BamHI 11 and SalI to prepare a DNA fragment containing thepresent soybean CPOX gene. The plasmid pBI121 KS constructed in Example9 was digested with the restriction enzymes BamHI and SalI to removeβ-glucuronidase gene and, instead thereof, the above DNA fragmentcontaining the present soybean CPOX gene was inserted therein toconstruct the plasmid pBISCPOX (FIG. 26) wherein the gene was ligated tothe downstream of 35S promoter.

The plasmid pBISCPOX was introduced into Agrobacterium tumefaciensLBA4404 and it was cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBISCPOX.

Tobacco leaf pieces cultured sterilely is infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco bearing the introduced present soybean CPOX gene is obtained.

EXAMPLE 44.

Production of Recombinant Tobacco Bearing Arabidopsis thalianaPPO(A220V) Gene and Present Soybean CPOX Gene

A plasmid is constructed for introducing both Arabidopsis thalianaPPO(A220V) gene and present soybean CPOX gene into a plant byAgrobacterium infection method. The plasmid pAPNSB constructed inExample 40 is digested with the restriction enzymes BamHI and DraI toisolate a DNA fragment of about 2.8 kb composed of Arabidopsis thalianaPPO(A220V) gene, the terminator of a nopaline synthase and 35S promoter.The fragment is inserted into the BamHI restriction site of the plasmidpBISCPOX constructed in Example 43. For selecting the plasmid pBIAPSCP(FIG. 27) wherein Arabidopsis thaliana PPO(A220V) gene and the presentson bean CPOX gene are ligated to the downstream of the 35S promoter,respectively, the resultant plasmid is digested with the restrictionenzymes NotI and SalI to select a clone producing a DNA fragment ofabout 2.0 kb composed of the 35S promoter and the present CPOX gene.

The plasmid pBIAPSCP is introduced into Agrobacterium tumefaciensLBA4404 and this is cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBIAPSCP.

Tobacco leaf pieces cultivated sterilely is infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco bearing the introduced both Arabidopsis thaliana PPO(A220V) geneand present soybean CPOX gene is obtained.

EXAMPLE 45

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Both Arabidopsis thaliana PPO(A220V) Gene and Present SoybeanCPOX Gene

The levels of resistance to herbicidal compounds are confirmedquantitatively by testing the tobacco bearing the introduced bothArabidopsis thaliana PPO(A220V) gene and present soybean CPOX geneproduced in Example 44 according to the same manner as in Example 37.

EXAMPLE 46

Isolation of Glyphosate Resistant Gene

Glyphosate resistant soybean (Glycine max) was seeded and cultivated at27

<C. for 30 days. The first leaves of germinated individuals werecollected, were frozen in liquid Nitrogen and were grounded in a mortarwith a pestle. Genomic DNA was extracted from the ground material with agenomic DNA extracting reagent ISOPLANT (manufactured by NIPPON GENE)according to the manual attached thereto. PCR was carried out by usingthe genomic DNA as a template, an oligonucleotide primer composed of thenucleotide sequence represented by SEQ ID NO: 72 and an oligonucleotideprimer composed of the nucleotide sequence represented by SEQ ID NO: 73to amplify a DNA fragment (hereinafter referred to as the presentCTP-CP4 EPSPS gene) containing a nucleotide sequence encoding achloroplast transit peptide sequence of EPSPS of petunia (Petuniahybrida) (hereinafter referred to as CTP) and EPSPS gene ofAgrobacterium (Agrobacterium sp. Strain CP4). The oligonucleotides weresynthesized by using a DNA synthesizer (PE Applied Biosystems: Model 394DNA/RNA Synthesizer) and purified with an oligonucleotide purificationcartridge (PE Applied Systems: OPS Cartridge). The PCR was carried outby maintaining at 94° C. for 5 minutes, 55° C. for 2 minutes and then at72° C. for 3 minutes, and further repeating a cycle for maintaining at94° C. for 1 minute, at 55° C. for 2 minutes and then 72° C. for 3minutes 38 times, and, finally, further maintaining at 94° C. for 1minute, at 55° C. for 2 minutes and then 72° C. for 10 minutes. Theamplified DNA fraction was ligated to a PCR product cloning site ofplasmid pCR2.1 (manufactured Invitrogen), to construct the plasmidpCREPSPS (FIG. 28). Then, the plasmid was introduced in a competent cellof E. coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) toselect an ampicillin resistant strain. The nucleotide sequence of theplasmid contained in the selected ampicillin resistant strain wasdetermined by using Thermo Sequence II Dye Terminator kit (manufactureby Amersham Pharmacia Biotech) and DNA Sequencer 373S (manufactured byPE Applied Biosystems). As a result, the nucleotide sequence representedby SEQ) ID NO: 74 was revealed and it was confirmed that the plasmidpCREPSPS contained the present CTP-CP4 EPSPS gene.

EXAMPLE 47

Introduction of Present CTP-CP4 EPSPS Gene into Tobacco

A plasmid was constructed for introducing the present CTP-CP4 EPSPS geneinto a plant by Agrobacterium infection method. First, pNG01 [Shiota etal., (1994) Plant Physiol., 106:17-23] (FIG. 29) was digested with therestriction enzyme HindIII and nucleotides were added to the gap of thedouble-stranded DNA with DNA Polymerase I to blunt the end of the DNA,followed by self-cyclization with T4 DNA ligase to obtain pNG04 (FIG.30). The plasmid pNG04 was digested with the restriction enzyme XbaI toobtain a DNA fragment containing the terminator of a nopaline synthaseand 35S promoter located at the downstream thereof. The fragment wasinserted in the XbaI restriction site of plasmid pUC19 (manufactured byTakara Shuzo Co., Ltd.) to obtain pNT35S (FIG. 31). Then, the plasmidpCREPSPS constructed in Example 46 was digested with the restrictionenzymes HindIII and SalI and the resultant DNA fragment containing thepresent CTP-CP4 EPSPS gene was inserted between HindIII and SalIrestriction sites of pNT35S to obtain the plasmid pCENS (FIG. 32). Theplasmid pCENS was digested with the restriction enzyme HindIII andnucleotides were added to the gap of the double-stranded DNA with DNAPolymerase I to blunt the end of the DNA. The 5′-end of the DNA wasdephosphorylated with treatment of alkaline phosphatase derived fromcalf small intestine, followed by insertion of phosphorylated KpnIlinker (4668A manufactured by Takara Shuzo Co., Ltd.) therein andcyclization to obtain the plasmid pCENSK (FIG. 33). The plasmid pBI121KSconstructed in Example 9 was digested with the restriction enzymes KpnIand SalI to remove β-glucuronidase gene and, instead thereof, a DNAfragment containing the present CTP-CP4 EPSPS gene, which was obtainedby digesting the above plasmid pCENSK with the restriction enzymes KpnIand SalI, was inserted therein to construct the plasmid pBICE (FIG. 34)wherein the present CTP-CP4 EPSPS gene was ligated to the downstream of35S promoter.

The plasmid pBICE was introduced into Agrobacterium tumefaciens LBA44044(manufactured by Clontech) and this was cultured in LB medium (0.5%yeast extract, 1.0% Bacto tryptone, 0.5% NaCl) containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBICE.

Tobacco leaf pieces cultivated sterilely were infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco bearing the inserted present CTP-CP4 EPSPS gene was obtained.

EXAMPLE 48

Introduction of Present CTP-CP4 EPSPS Gene and Gene Encoding VariantTobacco Chelatase Subunit Into Tobacco

A plasmid was constructed for introducing the present CTP-CP4 EPSPS geneand a gene encoding a variant tobacco chelatase subunit into a plant byAgrobacterium infection method. First, the plasmid pCENSK constructed inExample 47 was digested with the restriction enzyme KpnI to obtain a DNAfragment containing the present CTP-CP4 EPSPS gene, a terminator of agene encoding nopaline synthase located at the downstream thereof and35S promoter located at the downstream of the terminator. This wasinserted into the KpnI restriction site of the plasmid pBITCHLHconstructed in Example 9 to construct the plasmid pBICETCH (FIG. 35)wherein the present CTP-CP4 EPSPS gene and the gene encoding the varianttobacco chelatase subunit were ligated to the downstream of 35Spromoter, respectively.

The plasmid pBICETCH was introduced into Agrobacterium tumefaciensLBA44044 and this was cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBICETCH.

Tobacco leaf pieces cultivated sterilely were infected with theAgrobacterium strain and, according to the same manner as described inExample 5, tobacco bearing the inserted present CTP-CP4 EPSPS gene andgene encoding the variant tobacco chelatase subunit was obtained.

EXAMPLE 49

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Present CTP-CP4 EPSPS Gene as Well as Tobacco BearingIntroduced Present CTP-CP4 EPSPS Gene and Gene Encoding Variant TobaccoChelatase Subunit

Leaves of the tobacco bearing the introduced present CTP-CP4 EPSPS geneproduced in Example 47, those of the control recombinant tobaccoobtained in Example 5 and those bearing the introduced present CTP-CP4gene and gene encoding the variant tobacco chelatase subunit produced inExample 48 are collected, and each leaf is divided into the right andleft equivalent pieces along the main vein. One of the pieces is treatedwith an aqueous solution containing 0.3 ppm PPO inhibitory-typeherbicidal compound of Structure 8, while to the other piece is nottreated with the compound. These leaf pieces are placed on MS mediumcontaining 0.8% agar and allowed to stand at room temperature for 7 daysin a light place. Then, each leaf piece is ground in 5 ml of 80% aqueousacetone solution in a mortar with a pestle to extract chlorophyll. Theextract is diluted 10 times with 80% aqueous acetone solution and theabsorbance is measured at 750 nm, 663 nm and 645 nm to calculate thetotal chlorophyll content according to the method described by MacknneyG., J. Biol. Chem. (1941) 140, p 315. The resistant level to theherbicidal compound tested is represented by the percentage of the totalchlorophyll content of the leave piece treated with the herbicidalcompound to that of untreated leaf piece.

Similarly, the tobacco bearing the introduced present CTP-CP4 EPSPSgene, the tobacco bearing the introduced both present CTP-CP4 EPSPS geneand gene encoding the variant tobacco chelatase subunit, and the controlrecombinant tobacco are treated with an aqueous solution containing 100ppm of a glyphosate to determine the resistant level to the glyphosate.The resistant level to the glyphosate is represented by the percentageof the total chlorophyll content of the leave piece treated with theglyphosate to that of untreated leaf piece.

EXAMPLE 50

Introduction of Present CTP-CP4 EPSPS Gene and Gene Encoding VariantSoybean PPO into Tobacco

A plasmid was constructed for introducing the present CTP-CP4 EPSPS geneand a gene encoding a variant soybean PPO into a plant by Agrobacteriuminfection method. According to the same manner as described in Example11, PCR was carried out by using an oligonucleotide primer composed ofthe nucleotide sequence represented by SEQ ID NO: 75, an oligonucleotideprimer composed of the nucleotide sequence represented by SEQ ID NO: 76,and the plasmid pSPPO-P constructed in Example 3 as a template toamplify a DNA fragment containing the gene encoding the variant soybeanPPO. Then, the plasmid pBI121K constructed in Example 9 was digestedwith restriction enzymes KpnI and SacI to remove β-glucuronidase geneand, instead thereof, a DNA fragment obtained by digesting the above DNAfragment containing the gene encoding the variant soybean PPO with therestriction enzymes KpnI and SacI was inserted therein to construct theplasmid pBIGMP (FIG. 36) wherein the gene was ligated to the downstreamof the 35S promoter.

Then, the plasmid pCENSK constructed in Example 47 is digested with therestriction enzyme KpnI to obtain a DNA fragment containing the presentCTP-CP4 EPSPS gene, the terminator of the gene encoding nopalinesynthase located at the downstream of the gene and the 35S promoterlocated at the downstream of the terminator, followed by insertion of itin the KpnI restriction site of the above plasmid pBIGMP to constructthe plasmid pBICEGMP (FIG. 37) wherein the present CTP-CP4 EPSPS geneand the gene encoding the variant soybean PPO are ligated to thedownstream of the 35S promoter, respectively.

The plasmid pBICEGMP is introduced into Agrobacterium tumefaciensLBA44044 and this is cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBICEGMP.

Tobacco leaf pieces cultivated sterilely are infected with theAgrobacterium strain and, according to the same manner as in Example 5,tobacco bearing the introduced present CTP-CP4 EPSPS gene and geneencoding the variant soybean PPO is obtained.

EXAMPLE 51

Confirmation of Resistance to Herbicidal Compound of Tobacco BearingIntroduced Present CTP-CP4 EPSPS Gene and Gene Encoding Variant SoybeanPPO

The levels of resistance to the PPO inhibitory-type herbicidal compoundrepresented by structure 8 are confirmed quantitatively by testing thetobacco bearing the introduced present CTP-CP4 EPSPS gene and geneencoding the variant soybean PPO obtained in Example 50, and the controlrecombinant tobacco obtained in Example 5 according to the same manneras in Example 49.

Further the levels of resistance to glyphosate are confirmedquantitatively by testing the tobacco bearing introduced the presentCTP-CP4 EPSPS gene and the gene encoding the variant soybean PPO and thecontrol recombinant tobacco according to the same manner as in Example49.

EXAMPLE 52

Introduction of Present CTP-CP4 EPSPS Gene and Gene Encoding VariantChlamydomonas reinhardtii PPO Into Tobacco

A plasmid was constructed for introducing the present CTP-CP4 EPSPS geneand a gene encoding a variant Chlamydomonas reinhardtii PPO into a plantby Agrobacterium infection method. The plasmid pTVCRP constructed inExample 16 was digested with the restriction enzymes BamHI and SacI toprepare a DNA fragment containing a gene encoding a variantChlamydomonas reinhardtii PPO. Binary vector pB1121 (manufactured byClontech) was digested with the restriction enzymes BamHI and SacI toremove β-glucuronidase gene and, instead thereof, the above DNA fragmentcontaining the gene encoding the variant Chlamydomonas reinhardtii PPOwas inserted therein to construct the plasmid pBICRP (FIG. 38) whereinthe gene was ligated to the downstream of the 35S promoter.

Then, the plasmid pBICRP is digested with the restriction enzyme BamHIand nucleotides are added to the gap of the double-stranded DNA with DNAPolymerase I to blunt the end of the DNA. The 5′-end of the DNA isdephosphorylated by treatment with an alkaline phosphatase derived fromcalf small intestine, followed by inserting a phosphorylated KpnI linker(4668A manufactured by Takara Shuzo Co., Ltd.) and cyclization to obtainplasmid pBICRPK. Then, the plasmid pCENSK constructed in Example 47 isdigested with the restriction enzyme KpnI to obtain a DNA fragmentcontaining the present CTP-CP4 EPSPS gene, the terminator of the geneencoding nopaline synthase located at the downstream of the presentCTP-CP4 EPSPS gene and the 35S promoter located at the downstream of theterminator. This is inserted in the KpnI restriction site of the aboveplasmid pBICRPK to construct the plasmid pBICECRP (FIG. 39) wherein thepresent CTP-CP4 EPSPS gene and the gene encoding the variantChlamydomonas reinhardtii PPO are ligated to the downstream of 35Spromoter, respectively.

The plasmid pBICERP is introduced into Agrobacterium tumefaciensLBA44044 and this is cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBICECRP.

Tobacco leaf pieces cultivated sterilely are infected with theAgrobacterium strain and, according to the same manner as described inExample 5, tobacco bearing the introduced present CTP-CP4 EPSPS gene andgene encoding the variant Chlamydomonas reinhardtii PPO is obtained.

EXAMPLE 53

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Present CTP-CP4 EPSPS Gene and Gene Encoding VariantChlamydomonas reinhardtii PPO

The levels of resistance to the above PPO inhibitory-type herbicidalcompound represented by Structure 8 are confirmed quantitatively bytesting the tobacco bearing the introduced present CTP-CP4 EPSPS geneand gene encoding the variant Chlamydomonas reinhardtii PPO obtained inExample 52 and the control recombinant tobacco obtained in Example 5according to the same manner as in Example 49.

Further the levels of resistance to glyphosate are confirmedquantitatively by testing the tobacco bearing the introduced presentCTP-CP4 EPSPS gene and gene encoding the variant Chlamydomonasreinhardtii PPO and the control recombinant tobacco according to thesame manner as in Example 49.

EXAMPLE 54

Introduction of Present CTP-CP4 EPSPS Gene and Gene EncodingChloroplast-Localized Type Ferrochelatase of Arabidopsis thaliana IntoTobacco

The plasmid pBIATF constructed in Example 39 is digested with therestriction enzyme BamHI and then nucleotides are added in the gap ofthe double-stranded DNA with DNA polymerase I to blunt the end of theDNA. The 5′-end of the DNA is dephosphorylated by treatment with analkaline phosphatase derived from calf small intestine, followed byinsertion of a phosphorylated KpnI linker (4668A manufactured by TakaraShuzo Co. Ltd.) and cyclization to obtain the plasmid pBIATFK. Then, theplasmid pCENSK constructed in Example 47 is digested with therestriction enzyme KpnI to obtain a DNA fragment containing the presentCTP-CP4 EPSPS gene, the terminator of the gene encoding nopalinesynthase located at the downstream of the present CTP-CP4 EPSPS gene andthe 35S promoter located at the downstream of the terminator. This isinserted in the KpnI restriction site of the above plasmid pBIATFK toconstruct the plasmid pBICEATF (FIG. 40) wherein the present CTP-CP4EPSPS gene and the gene encoding the chloroplast-localized typeferrochelatase of Arabidopsis thaliana are ligated to the downstream of35S promoter, respectively.

The plasmid pBICEATF is introduced into Agrobacterium tumefaciensLBA44044 and this is cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBICEATF.

Tobacco leaf pieces cultivated sterilely are infected with theAgrobacterium strain and, according to the same manner as described inExample 5, tobacco bearing the inserted present CTP-CP4 EPSPS gene andgene encoding the chloroplast-localized type ferrochelatase ofArabidopsis thaliana is obtained.

EXAMPLE 55

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Present CTP-CP4 EPSPS Gene and Gene EncodingChloroplast-Localized Type Ferrochelatase of Arabidopsis thaliana

The levels of resistance to the above PPO inhibitory-type herbicidalcompound represented by Structure 8 are confirmed quantitatively bytesting the tobacco bearing the introduced present CTP-CP4 EPSPS geneand gene encoding the chloroplast-localized type ferrochelatase ofArabidopsis thaliana obtained in Example 54, and the control recombinanttobacco obtained in Example 5 according to the same manner as in Example49.

Further the levels of resistance to glyphosate are confirmedquantitatively by testing the tobacco bearing the introduced presentCTP-CP4 EPSPS gene and gene encoding the chloroplast-localized typeferrochelatase of Arabidopsis thaliana and the control recombinanttobacco according to the same manner as in Example 49.

EXAMPLE 56

Introduction of Present CTP-CP4 EPSPS Gene and Gene Encoding PresentSoybean CPOX into Tobacco

A plasmid was constructed for introducing the present CTP-CP4 EPSPS geneand a gene encoding the present soybean CPOX into a plant byAgrobacterium infection method. First, the plasmid pCRSCPOX constructedin Example 42 was digested with the restriction enzyme BamHI to preparea DNA fragment containing a gene encoding the present soybean CPOX. TheDNA fragment was inserted in the BamHI restriction site of the plasmidpBI121KS constructed in Example 9 to obtain the plasmid pBISCPOXGUS.This plasmid was digested with the restriction enzyme SalI to removeβ-glucuronidase gene, followed by self-cyclization to construct theplasmid pBISCPOX (FIG. 41) wherein the gene was ligated to thedownstream of the 35S promoter.

Then, the plasmid pBISCPOX is digested with the restriction enzyme BamHIand nucleotides are added to the gap of the double-stranded DNA with DNApolymerase I to blunt the end of the DNA. The 5′-end of the DNA isdephosphorylated by treatment with an alkaline phosphatase derived fromcalf small intestine, followed by inserting a phosphorylated KpnI linker(4668A manufactured by Takara Shuzo Co., Ltd.) therein and cyclizationto obtain the plasmid pBISCPOXK. Then, the plasmid pCENSK constructed inExample 47 is digested with the restriction enzyme KpnI to obtain a DNAfraction containing the present CTP-CP4 EPSPS gene, the terminator ofthe gene encoding nopaline synthase located at the downstream of thepresent CTP-CP4 EPSPS gene and the 35S promoter located at thedownstream of the terminator. This is inserted in the KpnI restrictionsite of the above plasmid pBISCPOXK to construct the plasmid pBICESCPOX(FIG. 42) wherein the present CTP-CP4 EPSPS gene and the gene encodingthe present soybean CPOX are ligated to the downstream of 35S promoter,respectively.

The plasmid pBICESCPOX is introduced into Agrobacterium tumefaciensLBA44044 and this is cultured in LB medium containing 300 μg/ml ofstreptomycin, 100 μg/ml of rifampicin and 25 μg/ml of kanamycin,followed by selection of a transformant to isolate an Agrobacteriumstrain bearing pBICESCPOX.

Tobacco leaf pieces cultivated sterilely are infected with theAgrobacterium strain and, according to the same manner as described inExample 45, tobacco bearing the inserted present CTP-CP4 EPSPS gene andgene encoding the present soybean CPOX is obtained.

EXAMPLE 57

Confirmation of Resistance to Herbicidal Compounds of Tobacco BearingIntroduced Present CTP-CP4 EPSPS Gene and Gene Encoding Present SoybeanCPOX

The levels of resistance to the above PPO inhibitory-type herbicidalcompound represented by Structure 8 are confirmed quantitatively bytesting the tobacco bearing the introduced present CTP-CP4 EPSPS geneand gene encoding the present soybean CPOX obtained in Example 56, andthe control recombinant tobacco according to the same manner as inExample 49.

Further the levels of resistance to glyphosate are confirmedquantitatively by testing the tobacco bearing the introduced presentCTP-CP4 EPSPS gene and gene encoding the present soybean CPOX and thecontrol recombinant tobacco according to the same manner as in Example49.

As described hereinabove, according to the present invention, weedcontrol compound-resistant plant can be produced.

1. A method for producing a transgenic plant that is resistant to a weed control compound, comprising the steps of: introducing into a plant cell a nucleotide sequence encoding a coproporphyrinogen III oxidase of Escherichia coli; expressing the nucleotide sequence; and regenerating the plant cell into a transgenic plant.
 2. The method according to claim 1, wherein the nucleotide sequence is operably ligated to a promoter sequence and a terminator sequence both of which are functional in the plant cell.
 3. A weed control compound-resistant plant produced by the method of claim
 1. 4. A method for controlling weeds comprising applying a weed control compound to a growth area comprising the plant of claim
 3. 5. A method for selecting a plant comprising applying a weed control compound to a growth area comprising the plant of claim 3 and other plants, and selecting either plant on the basis of difference in growth between the plants.
 6. A method for selecting a plant cell which comprises: applying a weed control compound to a plant cell transformed with a nucleotide sequence encoding: a coproporphyrinogen III oxidase of Escherichia coli and other plant cells; and selecting either plant cell on the basis of a difference in growth between the plant cells.
 7. A method for producing a transgenic plant that is resistant to a weed control compound inhibiting porphyrin biosynthesis, comprising the steps of: introducing into a plant cell a nucleotide sequence encoding a coproporphyrinogen III oxidase of Escherichia coli; expressing the nucleotide sequence; and regenerating the plant cell into a transgenic plant.
 8. A method for producing a transgenic plant that is resistant to a protoporphyrinogen IX oxidase inhibitory-type herbicidal compound, comprising the steps of: introducing into a plant cell a nucleotide sequence encoding a coproporphyrinogen III oxidase of Escherichia coli; expressing the nucleotide sequence; and regenerating the plant cell into a transgenic plant. 